Dataset Information

Complete Genomic and Lysis-Cassette Characterization of the Novel Phage, KBNP1315, which Infects Avian Pathogenic Escherichia coli (APEC).

ABSTRACT: Avian pathogenic Escherichia coli (APEC) is a major pathogen that causes avian colibacillosis and is associated with severe economic losses in the chicken-farming industry. Here, bacteriophage KBNP1315, infecting APEC strain KBP1315, was genomically and functionally characterized. The evolutionary relationships of KBNP1315 were analyzed at the genomic level using gene (protein)-sharing networks, the Markov clustering (MCL) algorithm, and comparative genomics. Our network analysis showed that KBNP1315 was connected to 30 members of the Autographivirinae subfamily, which comprises the SP6-, T7-, P60-, phiKMV-, GAP227- and KP34-related groups. Network decomposition suggested that KBNP1315 belongs to the SP6-like phages, but our comparison of putative encoded proteins revealed that key proteins of KBNP1315, including the tail spike protein and endolysin, had relative low levels of amino acid sequence similarity with other members of the SP6-like phages. Thus KBNP1315 may only be distantly related to the SP6-like phages, and (based on the difference in endolysin) its lysis mechanism may differ from theirs. To characterize the lytic functions of the holin and endolysin proteins from KBNP1315, we expressed these proteins individually or simultaneously in E. coli BL21 (DE3) competent cell. Interestingly, the expressed endolysin was secreted into the periplasm and caused a high degree of host cell lysis that was dose-dependently delayed/blocked by NaN3-mediated inhibition of the SecA pathway. The expressed holin triggered only a moderate inhibition of cell growth, whereas coexpression of holin and endolysin enhanced the lytic effect of endolysin. Together, these results revealed that KBNP1315 appears to use a pin-holin/signal-arrest-release (SAR) endolysin pathway to trigger host cell lysis.

SUBMITTER: Lee JS

PROVIDER: S-EPMC4640515 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Complete Genomic and Lysis-Cassette Characterization of the Novel Phage, KBNP1315, which Infects Avian Pathogenic Escherichia coli (APEC).

Lee Jung Seok JS Jang Ho Bin HB Kim Ki Sei KS Kim Tae Hwan TH Im Se Pyeong SP Kim Si Won SW Lazarte Jassy Mary S JM Kim Jae Sung JS Jung Tae Sung TS

PloS one 20151110 11

Avian pathogenic Escherichia coli (APEC) is a major pathogen that causes avian colibacillosis and is associated with severe economic losses in the chicken-farming industry. Here, bacteriophage KBNP1315, infecting APEC strain KBP1315, was genomically and functionally characterized. The evolutionary relationships of KBNP1315 were analyzed at the genomic level using gene (protein)-sharing networks, the Markov clustering (MCL) algorithm, and comparative genomics. Our network analysis showed that KBN ...[more]

PMID: 26555076

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Project description:Many reports show an association between the Pst system, the Pho regulon related genes and bacterial virulence. Our previous results showed that a functional Pst system is required for full virulence, resistance to serum, polymyxin B and acid shock However, the interplay between the Pst system and virulence has an unknown molecular basis. To understand global APEC virulent strain responses to Pho regulon activation, we conducted transcriptome profiling experiments comparing the isogenic Pst mutant with its wild type strain chi7122 using the Affymetrix GeneChipÂ® E. coli Genome 2.0 Array. To understand the global responses to Pho regulon activation of APEC, we use the Affymetrix technology. We conducted transcriptome profiling experiments comparing the APEC chi7122 strain and its isogenic Pst mutant grown in rich phosphate medium using the Affymetrix GeneChipÂ® E. coli Genome 2.0 Array. The Affymetrix GeneChipÂ® E. coli Genome 2.0 Array contains the genome of the E. coli MG1655 and three pathogenic E. coli strain (EDL933, Sakai and CFT073) representing 20,366 genes. While comparing genes expression between Pst mutant and the wild type chi7122 strain, 471 genes are either up- (254) or down-regulated (217) of at least 1.5-fold, with a p-value inferior or equal to 0.05 and a false discovery rate of 2.71%. Experiment Overall Design: RNA extraction was performed onto the chi7122 and K3 strains on four biological replicates. An overnight culture grown at 37Â°C was diluted 100-fold into 5 ml of LB broth and was allowed to grow to mid-log phase (OD600 0,6). Cultures were centrifuged and RNAs were isolated by using the RiboPureâ¢-Bacteria Kit (Ambion, Austin, TX), according to the manufacturerâs recommendations, with the exception that the DNAse 1 treatment was performed twice. RNA extractions were performed on four different days (the chi7122 and K3 RNA were extracted at the same time) and a total of eight hybridizations were performed. Hybridization was carried out at McGill University & Genome Quebec Innovation Centre according to the Affymetrix recommendation (Affymetrix Expression Manual Section 3 701029 rev. 4). GeneChip scan was carried out at McGill University & Genome Quebec Innovation Centre according to the Affymetrix recommendation (Affymetrix Expression Manual Section 3 701029 rev. 4), and data were processed using the FlexArray software (Michal Blazejczyk, Mathieu Miron, Robert Nadon (2007). FlexArray: A statistical data analysis software for gene expression microarrays. Genome Quebec, Montreal, Canada). Raw data were normalized using the RMA algorithm and log2 were generated. The expression value was generated by subtracting the mean value of each replicate of the mutant strain by wild-type strain.

Project description:BackgroundAvian pathogenic Escherichia coli (APEC) is a major cause of disease impacting animal health. The bone marrow is the reservoir of immature immune cells; however, it has not been examined to date for gene expression related to developmental changes (cell differentiation, maturation, programming) after APEC infection. Here, we study gene expression in the bone marrow between infected and non-infected animals, and between infected animals with mild (resistant) versus severe (susceptible) pathology, at two times post-infection.ResultsWe sequenced 24 bone marrow RNA libraries generated from the six different treatment groups with four replicates each, and obtained an average of 22 million single-end, 100-bp reads per library. Genes were detected as differentially expressed (DE) between APEC treatments (mild pathology, severe pathology, and mock-challenged) at a given time point, or DE between 1 and 5 days post-infection (dpi) within the same treatment group. Results demonstrate that many immune cells, genes and related pathways are key contributors to the different responses to APEC infection between susceptible and resistant birds and between susceptible and non-challenged birds, at both times post-infection. In susceptible birds, lymphocyte differentiation, proliferation, and maturation were greatly impaired, while the innate and adaptive immune responses, including dendritic cells, monocytes and killer cell activity, TLR- and NOD-like receptor signaling, as well as T helper cells and many cytokine activities, were markedly enhanced. The resistant birds' immune system, however, was similar to that of non-challenged birds.ConclusionThe DE genes in the immune cells and identified signaling models are representative of activation and resolution of infection in susceptible birds at both post-infection days. These novel results characterizing transcriptomic response to APEC infection reveal that there is combinatorial activity of multiple genes controlling myeloid cells, and B and T cell lymphopoiesis, as well as immune responses occurring in the bone marrow in these early stages of response to infection.

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Project description:BACKGROUND: Avian pathogenic E. coli (APEC) are associated with extraintestinal diseases in poultry. The pstSCAB-phoU operon belongs to the Pho regulon and encodes the phosphate specific transport (Pst) system. A functional Pst system is required for full virulence in APEC and other bacteria and contributes to resistance of APEC to serum, to cationic antimicrobial peptides and acid shock. The global mechanisms contributing to the attenuation and decreased resistance of the APEC pst mutant to environmental stresses have not been investigated at the transcriptional level. To determine the global effect of a pst mutation on gene expression, we compared the transcriptomes of APEC strain chi7122 and its isogenic pst mutant (K3) grown in phosphate-rich medium. RESULTS: Overall, 470 genes were differentially expressed by at least 1.5-fold. Interestingly, the pst mutant not only induced systems involved in phosphate acquisition and metabolism, despite phosphate availability, but also modulated stress response mechanisms. Indeed, transcriptional changes in genes associated with the general stress responses, including the oxidative stress response were among the major differences observed. Accordingly, the K3 strain was less resistant to reactive oxygen species (ROS) than the wild-type strain. In addition, the pst mutant demonstrated reduced expression of genes involved in lipopolysaccharide modifications and coding for cell surface components such as type 1 and F9 fimbriae. Phenotypic tests also established that the pst mutant was impaired in its capacity to produce type 1 fimbriae, as demonstrated by western blotting and agglutination of yeast cells, when compared to wild-type APEC strain chi7122. CONCLUSION: Overall, our data elucidated the effects of a pst mutation on the transcriptional response, and further support the role of the Pho regulon as part of a complex network contributing to phosphate homeostasis, adaptive stress responses, and E. coli virulence.

Dataset Information

Complete Genomic and Lysis-Cassette Characterization of the Novel Phage, KBNP1315, which Infects Avian Pathogenic Escherichia coli (APEC).

Publications

Complete Genomic and Lysis-Cassette Characterization of the Novel Phage, KBNP1315, which Infects Avian Pathogenic Escherichia coli (APEC).

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