Cloning, purification, and functional characterization of Carocin S2, a ribonuclease bacteriocin produced by Pectobacterium carotovorum

<p>Abstract</p> <p>Background</p> <p>Most isolates of <it>Pectobacterium carotovorum subsp. carotovorum </it>(Pcc) produce bacteriocins. In this study, we have determined that Pcc strain F-rif-18 has a chromosomal gene encoding the low-molecular-weight bacteriocin, Carocin S2, and that this bacteriocin inhibits the growth of a closely related strain. Carocin S2 is inducible by ultraviolet radiation but not by mutagenic agents such as mitomycin C.</p> <p>Results</p> <p>A carocin S2-defective mutant, TF1-2, was obtained by Tn5 insertional mutagenesis using F-rif-18. A 5706-bp DNA fragment was detected by Southern blotting, selected from a genomic DNA library, and cloned to the vector, pMS2KI. Two adjacent complete open reading frames within pMS2KI were sequenced, characterized, and identified as caroS2K and caroS2I, which respectively encode the killing protein and immunity protein. Notably, carocin S2 could be expressed not only in the mutant TF1-2 but also in <it>Escherichia coli </it>DH5α after entry of the plasmid pMS2KI. Furthermore, the C-terminal domain of CaroS2K was homologous to the nuclease domains of colicin D and klebicin D. Moreover, SDS-PAGE analysis showed that the relative mass of CaroS2K was 85 kDa and that of CaroS2I was 10 kDa.</p> <p>Conclusion</p> <p>This study shown that another nuclease type of bacteriocin was found in <it>Pectobacterium carotovorum</it>. This new type of bacteriocin, Carocin S2, has the ribonuclease activity of CaroS2K and the immunity protein activity of CaroS2I.</p

Serial increase of IL-12 response and human leukocyte antigen-DR expression in severe sepsis survivors

Introduction: Sepsis-induced immunosuppression may result in death. The mechanisms of immune suppression include loss of macrophage and monocyte expression of the major histocompatibility complex, increased anti-inflammatory cytokine expression and decreased expression of proinflammatory cytokines. In this study, we sought to determine the mechanisms of immune suppression in severe sepsis by repeated detection. Methods: We designed this prospective observational study to measure monocyte human leukocyte antigen (HLA)-DR expression, plasma cytokine levels and cytokine responses on days 1 and 7 in stimulated peripheral blood mononuclear cells (PBMCs) of healthy controls and patients with severe sepsis. Results: Of the 35 enrolled patients, 23 survived for 28 days and 12 died, 6 of whom died within 7 days. Plasma levels of IL-1 beta, IL-6, IL-10, IL-17, transforming growth factor (TGF)-beta 1 and TNF-alpha were higher, but plasma IL-12 level was lower in septic patients than those in controls. Day 1 plasma levels of IL-1 beta, IL-6, IL-10 and TGF-beta 1 in nonsurvivors were higher than those in survivors. Day 7 plasma IL-10 levels in nonsurvivors were higher than in survivors. IL-1 beta response was higher, but IL-12 and TNF-alpha responses were lower in septic patients than in controls. Day 1 IL-6 response was lower, but day 1 TGF-beta 1 response was higher in nonsurvivors than in survivors. Plasma IL-6 and IL-10 levels were decreased in survivors after 6 days. IL-6 response was decreased in survivors after 6 days, but IL-12 response was increased. Monocyte percentage was higher, but positive HLA-DR percentage in monocytes and mean fluorescence intensity (MFI) of HLA-DR were lower in septic patients than in controls. MFI of HLA-DR was increased in survivors after 6 days. Conclusions: Monocyte HLA-DR expression and IL-12 response from PBMCs are restored in patients who survive severe sepsis

Studies of Functional Characterization and Secretion Mechanism of Low-Molecule-Weight bacteriocin from Pectobacterium carotovorum subsp. carotovorum

Pectobacterium carotovorum subsp. carotovorum 是一株屬於腸道菌科的革蘭氏陰性菌，舊稱為伊文氏桿菌 (Erwinia)。這種革蘭氏陰性菌偏好寄生於植物，它經常造成經濟作物損傷。被此細菌寄生的植物在高溫潮溼的環境下會產生根部腐爛的疾病，所以這種有名的植物致病菌一直以來是許多科學家致力研究重點之一。現行針對此種植物致病菌經常會採用以化學物質為主成分的抗菌藥劑進行防疫，但這些化學試劑並不是一種很有效的方法，原因是化學試劑的影響是全面的並沒有針對性。這些結果可能會影響到植栽區域的正常生態鏈，以及無法避免的環境污染。 無論是革蘭氏陽性或者是革蘭氏陰性菌都會分泌許多胞外蛋白質性物質，其中一種胞外蛋白質稱為細菌素。細菌素是一種由蛋白質組成的抗菌性毒性物質。此類毒性物質被生產者利用來抑制其他菌株的生存。然而這種蛋白質性的毒性物對於其所抑制菌株的種類是相當有限且有其特異性。換句話說此類具有攻擊特異性的蛋白質性物質或許是一種可以利用來有效防疫 Pectobacterium 所造成的損失。同時蛋白質性物質不易造成環境污染，故或許可以利用細菌素來做為一種不錯的生物性抗菌試劑。目前已經有許多文獻探討過這種蛋白質性物質，例如大腸桿菌所產生的大腸桿菌素以及綠膿桿菌所產生的綠膿桿菌素都是相當著名的細菌素。本篇論文即介紹我們在伊文氏桿菌也成功得選殖出低蛋白分子量的細菌素基因，並且將此類低分子量細菌素命名為 Carocin。同時我們也發現主司 Carocin 分泌機制的分泌系統為革蘭氏陰性菌上常見的第三類型分泌系統。雖然許多革蘭氏陰性菌皆保有此種分泌系統，但是過去卻沒有相關文獻證實過第三類分泌系統會參與細菌素的分泌。 本篇論文利用細菌的接合生殖將帶有抗藥性基因的 DNA 分子送入待研究的細菌素生產者細胞內進行非特定的基因阻斷。在此處我們選用本身帶有的轉位子 Tn5 的菌株 E. coli 1830 來當做接合生殖實驗的提供者，而待研究的伊文氏桿菌則為轉位子的接收者，期望能得到帶有細菌素相關基因被轉位子阻斷的突變株。另外對於特定基因的阻斷，則是利用同質互換的實驗方法來得到特定基因阻斷的突變株。這些被阻斷的基因則是用不對稱交聯聚合酵素鏈鎖反應 （Thermal asymmetric interlaced PCR） 解析其 DNA 序列。並且同時利用南方點墨法來驗證其結果，而南方點墨法亦利用於製備 Genomic DNA library，幫助我們可以得到直接由染色體上截取特定區域的 DNA 分子。北方點墨法與 RNA 逆轉錄實驗則是用來觀察實驗中突變株細胞內的基因轉錄情形。 藉由轉位子 Tn5 阻斷基因的實驗方法，我們找到並且已經發表了兩個由伊文氏桿菌所生產的低分子量細菌素。 Carocin S1 為第一個發表的低分子量細菌素，它是一種由 Pcc 菌株 H-rif-8-6 所產生，可將 DNA 水解的核酸水解型細菌素。另外一個由 F-rif-18 產生的細菌素則命名為 Carocin S2。 經由核酸水解實驗， Carocin S2 被證實為一種可以水解 RNA 分子的細菌素，但令我們感興趣的是此種細菌素不但可以水解相對分子量最大的核糖體 RNA 也可水解其它小分量的 RNA。我們推測不論是 Carocin S1，亦或是 Carocin S2 在感染其他細菌細胞後，皆會攻擊它們所辨識的 DNA 或 RNA 分子，並進行水解破壞，使基因表現不正常，進而導致細菌細胞凋亡。而同時生產者本身亦會表現免疫蛋白來保護自己免於被自己生產的細菌素 Carocin 所傷害。另外，我們也發現伊文氏桿菌是藉由第三類分泌系統的鞭毛型構造，將毒性蛋白 Carocin 運輸至胞外，進而攻擊其它細菌細胞。 本篇論文將闡述我們實驗室發現了兩個由 Pectobacterium 產生的核酸水解型的低分子量細菌素 (Carocin S1 與 Carocin S2)，此種細菌素經常需要 UV 照射誘導使其表現。另外我們也證實此類細菌素是由鞭毛型第三類型分泌系統來輔助其分泌。Pectobacterium carotovorum subsp. carotovorum is a Gram-negative, phytoparasitic enterobacterium. It is also a well-known phytopathogen causing soft-rot disease of many economic crops. Chemical bactericidal is a current agent used against the disease but unavoidably causes the environmental contamination. Bacteriocins are endogenous, antimicrobial and toxic proteins, which are usually produced by Gram-positive and Gram-negative bacteria. However, the proteinaceous toxins have narrow spectrum to inhibit growth of the related bacteria; that is, bacteriocins would be a eco-friendly and efficient choice to prevent pathogen that causes the economic damage. While bacteriocins have been extensively investigated in many Gram-negative bacteria such as pyocin of Pseudomonas and colicin of Escherichia coli, they have been relatively unexplored in Pectobacterium species. The dissertation described that the Pcc strain also produces bacteriocin, designated as Carocin. Furthermore, we showed the secretion dependency of Carocin from Pcc strain used the type III secretion system, whereas little is shown about the relationship between them. In this study, the bacterial conjugation and the homologous replacement method were performed to translocate a linear construct harboring the antibiotic-resistance gene into the carocin-producing cell, resulting in the carocin-related null alleles. E. coli 1830 strain harboring transposon Tn5 was used as donors while the recipients were Pcc strains. In contrast to the conjugation, the homologous replacement method was used to knock out the specific target gene in genomic DNA. By using the thermal asymmetric interlaced PCR, the interrupted DNA sequence of the carocin-related null allele was determined. The southern blotting was used to confirm the result of mutation, moreover, the method would be used to prepare the genomic DNA library from which the native carocin-contained DNA was obtained. Additionally, transcriptional analysis was carried out by the Northern blotting and the reverse transcription PCR. These methods provide more information concerning with the carocins. Consequently, it was found that the Carocin S1 was produced from a Pcc strain H-rif-8-6. Carocin S1 has nucleotidase activity against DNA molecule. This is the first bacteriocin determining from Pectobacterium. Subsequently, Carocin S2 was characterized from Pcc strain F-rif-18, which was a RNase type bacteriocin. In the in vitro RNA degradation assay, Carocin S2 would hydrolyze not only the large molecule of ribosomal RNA but small RNA molecules. We suggested that both Carocin S1 and Carocin S2 kill those susceptive cells by exhausting their supply of DNA or RNA respectively, and then leading to inactivation of physiological biosynthesis. The two producers also have expression of the cognate immunity proteins which protect themselves from the specific damage of their toxic Carocins. Eventually, we established that Carocin protein might be secreted though the flagella that belongs type III secretion system. This secretion mechanism was different from those of previous reports of other bacteriocins. Here we showed the first two low-molecule-weight bacteriocins, Carocin S1 and Carocin S2, which are produced by Pectobacterium after UV irradiation. Furthermore, we found that the Carocin secretion is dependent on the type III secretion system integral to the bacterial flagellum, which this finding is irrelevant to the previous studies of bacteriocin secretion.Abstract 中文摘要.......................................................................................... i Abstract................................................................................................ iii Chapter 1 General introduction........................................................... 1 Features of Pectobacterium carotovorum subsp. carotovorum.............. 2 Features of bacteriocin........................................................................ 2 Induction of bacteriocin....................................................................... 4 Importation of bacteriocin.................................................................... 5 Killer domain of bacteriocin................................................................. 6 Secretion of bacteriocin......................................................................... 7 Summary............................................................................................... 10 Chapter 2 Cloning and Expression of the Erwinia carotovora subsp. carotovora Gene Encoding the Low-Molecular-Weight Bacteriocin Carocin S1......................................................................................................... 11 Introduction........................................................................................... 12 Materials and methods.......................................................................... 12 Results................................................................................................... 18 Discussion.............................................................................................. 20 Figures................................................................................................... 23 Chapter 3 Cloning, purification, and functional characterization of Carocin S2, a ribonuclease bacteriocin produced by Pectobacterium carotovorum............................. 33 Introduction............................................................................................ 34 Materials and methods................................................................................................. 34 Results................................................................................................... 43 Discussion.............................................................................................. 48 Figures................................................................................................... 51 Chapter 4 Extracellular secretion of Carocin S1 in Pectobacterium carotovorum subsp. carotovorum occurs via the type III secretion system integral to the bacterial flagellum............................................................................................... 67 Introduction........................................................................................... 68 Materials and methods.......................................................................... 68 Results................................................................................................... 76 Discussion.............................................................................................. 80 Figures................................................................................................... 83 Chapter 5 Conclusion............................................................................. 96 Conclusion.............................................................................................. 97 Figures...................................................................................................103 Tables Table 1. Bacteria and plasmids used in the study.................................105 Table 2. Primers used in this study.......................................................106 Table 3. The thermal processes of TAIL-PCR..........................................107 References.............................................................................................10

Low-Dose Steroid Therapy Is Associated with Decreased IL-12 Production in PBMCs of Severe Septic Patients

Background. Sepsis-induced immunosuppression may result in higher mortality rates in patients. Methods. We examined the relationship of cytokine responses from stimulated peripheral blood mononuclear cells (PBMCs) and monocyte human leukocyte antigen-DR (HLA-DR) expression (days 1 and 7) with low-dose steroid therapy in 29 septic patients. Patients were treated according to the guidelines. Thirty healthy controls were enrolled for validation. Results. Eighteen patients were prescribed low-dose steroids and 11 were not. Interleukin- (IL-) 12 responses in patients without low-dose steroid therapy on days 1 and 7 were higher than those with low-dose steroid therapy. Compared to day 1, IL-12 responses significantly increased on day 7 in patients without low-dose steroid therapy. After regression analysis, the change in the IL-12 response from day 7 to day 1 was found to be independently associated with the low-dose steroid therapy. There was no difference in monocyte HLA-DR expression between patients treated with and without low-dose steroid on day 1 or 7. No change in monocyte HLA-DR expression from day 7 to day 1 was observed in patients with or without low-dose steroid therapy. Conclusion. Decreased IL-12 response was associated with the low-dose steroid therapy in PBMCs of septic patients

Diguanylate Cyclase (DGC) Implicated in the Synthesis of Multiple Bacteriocins via the Flagellar-Type III Secretion System Produced by <i>Pectobacterium carotovorum</i> subsp. <i>carotovorum</i>

The plant pathogen Pectobacterium carotovorum subsp. carotovorum (previously Erwinia carotovora subsp. carotovora) causes soft rot and stem rot diseases in a variety of crops, including Chinese cabbage, potato, and tomato. The flagellar-type III secretion systems were used by Pcc’s virulence mechanism to export proteins or bacteriocins to the outside of the cell. DGC, a virulence factor that cyclizes c-di-GMP, a common secondary signal in physiological processes and toxin control systems of many bacteria, was discovered in Pcc’s genomic DNA. The dgc gene in Pcc was blocked using the method of homologous recombination in our study. In the in vivo setting, the results demonstrated that the dgc knockout strain does not release low molecular weight bacteriocins. The bacteriocin gene (carocin S2, carocin S3, carocin S4) and the flagellar-type III secretion system genes were also unable to be transcribed by the dgc knockout strain in the transcription experiment. We also observed that the amount of bacteriocin expressed changed when the amount of L-glutamine in the environment exceeded a particular level. These data suggested that L-glutamine influenced physiological processes in Pcc strains in some way. We hypothesized a relationship between dgc and the genes involved in Pcc LMWB external export via the flagellar-type secretion system based on these findings. In this study, the current findings led us to propose a mechanism in which DGC’s cyclic di-GMP might bind to receptor proteins and positively regulate bacteriocin transcription as well as the synthesis, mobility, and transport of toxins