Influence of environmental conditions on infection of Klebsiella pneumoniae by two different types of bacteriophages

The adsorption and efficiency of plating of bacteriophages FC3-1 and FC3-9 on Klebsiella pneumoniae C3 (serotype O1:K66) cells grown at different pHs and temperatures were quantitated. Bacteriophage FC3-1, with lipopolysaccharide as its bacterial receptor, showed a large decrease in efficiency of plating on bacteria grown at low pH or low temperature. Under the same conditions, no significant decrease in efficiency of plating was found for bacteriophage FC3-9, a phage requiring capsule and lipopolysaccharide for its adsorption and carrying capsule-depolymerizing activity. We demonstrate that K. pneumoniae C3 cells grown at low pH or low temperature have less lipopolysaccharide exposed on their surface. We conclude that this is why lipopolysaccharide-specific phage FC3-1 less efficiently infects bacterial cells grown under those conditions. We propose that bacteriophage FC3-9 efficiently infects bacterial cells grown at low pH or low temperature because its enzymatic activity on the capsule makes lipopolysaccharide available to this phage.     

Lipopolysaccharide-specific bacteriophage for Klebsiella pneumoniae C3.

Bacteriophage FC3-1 is one of several specific bacteriophages of Klebsiella pneumoniae C3 isolated in our laboratory. Unlike receptors for other Klebsiella phages, the bacteriophage FC3-1 receptor was shown to be lipopolysaccharide, specifically the polysaccharide fraction (O-antigen and core region). We concluded that capsular polysaccharide, outer membrane proteins, and lipid A were not involved in phage binding. Mutants resistant to this phage were isolated and were found to be devoid of lipopolysaccharide O-antigen by several criteria but to contain capsular material serologically identical to that of the wild type. The polysaccharide fraction was concluded to be the primary phage receptor, indicating that it is available to the phage.     

Isolation and characterization of bacteriophage FC3-10 from Klebsiella spp.

FC3-10 is a Klebsiella spp. specific bacteriophage isolated on a rough mutant (strain KT707, chemotype Rd) of K. pneumoniae C3. The bacteriophage receptor for this phage was shown to be the low-molecular mass lipopolysaccharide (LPS) fraction (LPS-core oligosaccharides), specifically the heptose content of the LPS inner-core. This is the first phage isolated on Klebsiella, the receptor for which is the LPS-core. This phage was unable to plate on Salmonella typhimurium LPS mutants with chemotypes Rd2 or Re showing incomplete or no heptose content on their LPS-core, respectively. Spontaneous phage-resistant mutants from different Klebsiella strains were deep-rough LPS mutants or encapsulated revertants from unencapsulated mutant strains.     

Coevolution of bacteriophage PP01 and Escherichia coli O157:H7 in continuous culture

The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h(-1) and by 3 orders of magnitude at a lower dilution rate (0.327 h(-1)). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h(-1) and persisted until the end of the experiment (approximately 200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture.     

Variation in the structure and bacteriophage-inactivating capacity of Salmonella anatum lipopolysaccharide as a function of growth temperature.

Growth temperature affects both the structure and the phage-inactivating capacity of Salmonella anatum A1 lipopolysaccharide. Whereas S. anatum cells normally synthesize smooth lipopolysaccharide when grown at physiological temperature (37 degrees C), a partial smooth-rough transition occurs when cells are grown at low temperature (20 to 25 degrees C). The synthesis at low growth temperature of lipopolysaccharide molecules lacking O-antigen was detected both by increased sensitivity of cells to the rough-specific bacteriophage Felix O-1 and by fractionation of oligosaccharides derived from lipopolysaccharide by mild acid hydrolysis. Growth temperature-induced changes in the structure of S. anatum A1 lipopolysaccharide also affected its ability to inactivate epsilon15, a bacteriophage that binds initially to the O-antigen portion of the molecule. Purified lipopolysaccharide prepared from cells grown at low growth temperature exhibited a higher in vitro phage-inactivating capacity than did lipopolysaccharide prepared from cells grown at physiological temperature (37 degrees C).     

Characterization of an O-antigen bacteriophage from Aeromonas hydrophila.

A unique bacteriophage of Aeromonas hydrophila serotype O:34 was isolated, purified, and characterized. The bacterial surface receptor was shown to be the O-antigen polysaccharide component of lipopolysaccharide specific to serotype O:34, which was chemically characterized. The high molecular weight lipopolysaccharide fraction (a fraction enriched in O antigen) was fully able to inactivate bacteriophage PM1. Phage-resistant mutants of A. hydrophila O:34 were isolated and found to be specifically devoid of lipopolysaccharide O antigen. No other cell-surface molecules were involved in phage binding. The host range of bacteriophage PM1 was found to be very narrow, producing plaques only on A. hydrophila strains from serotype O:34.     

Bacteriophage P22 H5 transfection and infection of plasmid Salmonella strains

The results of the Ca2+-dependent transfection of the DNA of bacteriophage P22 H5 to constructed Salmonella typhimurium F'- and R+-strains LT2 WT-R and SA118 demonstrated that in these salmonellae the effectiveness of transfection depended on the specificity of the interrelation of plasmids with host strains. Plasmids RA1, R538-1 and RP1 stimulated the transfection of S. typhimurium strain LT2 WT-R, but suppressed the transfection ability of S. typhimurium strain SA118. At the same time the expression of the function of plasmids R446b and R64-11 did not depend on the host strain, as the former did not affect and the latter suppressed the release of transfectants in both Salmonella strains. The presence of plasmids R124, RA1, R64-11 and R724 in strain SA118, heat-sensitive in respect to the synthesis of cell-wall lipopolysaccharide, not only led to a decrease in the effectiveness of transfection; the effectiveness of the inoculation of bacteriophage P22 H5 was also suppressed 10(4) times in the presence of plasmid R124 and at least 10(10) times in the presence of 3 other plasmids. The development of resistance to S-specific bacteriophage P22 H5 was not linked with disturbances in the adsorption of this bacteriophage. Besides, the addition of CaCl2 into the medium completely removed the limitation of infection with bacteriophage P22 H5, determined by plasmid R124.     

Mutations in the waaR gene of Escherichia coli which disrupt lipopolysaccharide outer core biosynthesis affect cell surface retention of group 2 capsular polysaccharides

On the basis of increased resistance to K5 capsule-specific bacteriophage, a waaR transposon mutant defective in the biosynthesis of lipopolysaccharide outer core was isolated. In a K1-expressing strain the mutation equally affected sensitivity to K1 capsule-specific bacteriophage, indicating a general effect on group 2 capsules. The waaR mutation affected retention on the cell surface of the K5 polysaccharide, with increased polysaccharide accumulating in the culture supernatant. This indicates that interactions between the outer core of lipopolysaccharide and group 2 capsular polysaccharides are important for the stabilization of group 2 capsular polysaccharides on the cell surface.     

Evaluation of the passage of Lactobacillus gasseri K7 and bifidobacteria from the stomach to intestines using a single reactor model

Background

The antibacterial activity of bacteriophages has been described rather well. However, knowledge about the direct interactions of bacteriophages with mammalian organisms and their other, i.e. non-antibacterial, activities in mammalian systems is quite scarce. It must be emphasised that bacteriophages are natural parasites of bacteria, which in turn are parasites or symbionts of mammals (including humans). Bacteriophages are constantly present in mammalian bodies and the environment in great amounts. On the other hand, the perspective of the possible use of bacteriophage preparations for antibacterial therapies in cancer patients generates a substantial need to investigate the effects of phages on cancer processes.

Results

In these studies the migration of human and mouse melanoma on fibronectin was inhibited by purified T4 and HAP1 bacteriophage preparations. The migration of human melanoma was also inhibited by the HAP1 phage preparation on matrigel. No response of either melanoma cell line to lipopolysaccharide was observed. Therefore the effect of the phage preparations cannot be attributed to lipopolysaccharide. No differences in the effects of T4 and HAP1 on melanoma migration were observed.

Conclusion

We believe that these observations are of importance for any further attempts to use bacteriophage preparations in antibacterial treatment. The risk of antibiotic-resistant hospital infections strongly affects cancer patients and these results suggest the possibility of beneficial phage treatment. We also believe that they will contribute to the general understanding of bacteriophage biology, as bacteriophages, extremely ubiquitous entities, are in permanent contact with human organisms.     

东方鲎C因子的分段克隆及表达

东方鲎C因子 (factorCfromTachypleustridentatus)是鲎血细胞中的一种对内毒素敏感的丝氨酸蛋白酶原 ,它与内毒素特异结合的特性 ,使之具有很大的应用价值。根据报道的C因子序列 ,设计了两对引物 ,以中国福建沿海的东方鲎 (Tachypleustridentatus)为材料抽提其血细胞总RNA ,首次用RT PCR的方法分两段扩增了鲎血中编码C因子蛋白的基因全序列。序列分析表明 ,得到的FC基因与文献报道的来自日本的东方鲎的FC有很高的同源性。将分段克隆得到的两段FC片段经相应酶切后 ,与含T7启动子的质粒 pET 2 8a( )在一个体系中作连接反应 ,构建表达质粒pET2 8a FC ,转化大肠杆菌BL2 1(DE3) ,筛选表达菌株。表达菌株经 1mmol/LIPTG诱导表达 2 .5h后 ,收集菌体并超声破菌。经SDS PAGE分析表明 ,在 110kD左右处有明显的表达条带 ,大小与FC的计算分子量相吻合。但表达产物以包涵体形式存在。经洗涤与变复性后 ,重组FC在体外表现出明显的抑菌活性。同时蛋白质印迹实验也提示了在大肠杆菌中FC的单基因表达物可能发生部分自剪切反应 ,而形成了额外的免疫印迹条带     

Isolation and characterization of bacteriophage PM2 from Aeromonas hydrophila

PM2 is an Aeromonas-specific bacteriophage isolated on A. hydrophila strain AH-3. The bacteriophage receptor for this phage was found to be the lipopolysaccharide (LPS), specifically a low-molecular weight LPS fraction (LPS-core oligosaccharides). Mutants resistant to this phage were isolated and found to be devoid of LPS O-antigen and altered in the LPS-core. No other outer-membrane (OM) molecules appeared to be involved in phage binding.     

The lipopolysaccharide core type of Escherichia coli O157:H7 and other non-O157 verotoxin-producing E. coli

A mouse monoclonal antibody specific for the R3 lipopolysaccharide core type of Escherichia coli was used to determine the core type of E. coli O157:H7 and other non-O157 verotoxin-producing E. coli strains. Lipopolysaccharide extracts from 28 clinical isolates were examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting and all were found to have the R3 core. None of the core lipopolysaccharide from the strains tested reacted with the control R1 and R2 specific monoclonal antibodies. A common core type between all the verotoxin-producing E. coli strains tested may be significant when considering the immune response to these bacteria, and to the receptor for the VT bacteriophage.     

Characteristics of Salmonella mutants competent for isolated nucleic acids

The method for the isolation of transfectable salmonellae by growing them in a liquid medium for a long time and selecting R-clones, subsequently tested in the Ca2+-dependent transfection of the DNA of bacteriophage P22 H5, was developed. The probability of obtaining transfectable clones varied between 18.3% and 48.3% in different Salmonella strains. Testing their sensitivity to specific bacteriophages used for the determination of structural distortions in the lipopolysaccharide layer of the cell wall made it possible to divide transfectable Salmonella clones into 3 phenotypical groups. The effectiveness of the formation of transfectants in the Ca2+-dependent transfection of the DNA of bacteriophage P22 H5 increased to 4.0 X 10(-8) - 8.0 X 10(-8) in the isolated R-clones of S. typhimurium strain SU453. In contrast to the initial strain, these R-clones were characterized not only by more effective transfection, but also by the possibility of plasmid transformation.     

Analysis of the phosphorylation level in guard-cell plasma membrane H+-ATPase in response to fusicoccin

A fungal phytotoxin fusicoccin (FC) causes irreversible opening of stomata by activation of the plasma membrane H+-ATPase in guard cells. However, the mechanism by which FC activates the H+-ATPase is not fully understood with respect to the event of phosphorylation. In this study, we provide quantitative evidence that FC-dependent activation of H+-ATPase requires the phosphorylation of the C-terminus, and that FC maintains the activated state by preventing the dephosphorylation. The plasma membrane H+-ATPase in guard cells was phosphorylated on serine and threonine residues in the C-termini of both VHA1 and VHA2 by FC, and the phosphorylation level paralleled the rates of H+-pumping and ATP hydrolysis. An endogenous 14-3-3 protein was co-precipitated with the H+-ATPase, and the amount of 14-3-3 protein was proportional to the phosphorylation level of H+-ATPASE: The recombinant 14-3-3 protein bound to the C-terminus only when it was phosphorylated, even in the presence of FC. The phosphorylated C-terminus was dephosphorylated by alkaline phosphatase, and the dephosphorylation was completely prevented when the C-terminus had been incubated with both FC and 14-3-3 protein. The results suggest that FC activates the H+-ATPase by accumulating the complex of phosphorylated H+-ATPase and 14-3-3 protein through inhibition of the dephosphorylation in guard cells.     

Coevolution of Bacteriophage PP01 and Escherichia coli O157:H7 in Continuous Culture

The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h⁻¹ and by 3 orders of magnitude at a lower dilution rate (0.327 h⁻¹). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h⁻¹ and persisted until the end of the experiment (~200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture.     

The properties of the Pseudomonas aeruginosa bacteriophage phiPMG1

The properties of the isolated Pseudomonas aeruginosa bacteriophage phiPMG1 include the lytic infection cycle, and the formation of a broad halo (semi-transparent zone) around the plaques. We consider phiPMG1 as a potential member of therapeutic cocktails of live phages, and as a source of peptidoglycan and lipopolysaccharide degrading enzymes. Partial sequencing of phiPMG1 genome has revealed high similarity with known temperate P. aeruginosa phage D3. An open reading frame encoding lytic transglycosilase was identified in the genome. This enzyme PMG MUR was obtained in recombinant form, and its activity and substrate specificity has been studied.     

A pilot study on the transcriptional response of androgen- and insulin-related genes in peripheral blood mononuclear cells induced by testosterone administration in hypogonadal men

The aim of the present study is to determine whether testosterone (T) administration changes the expression profile of androgen- and insulin-related genes in peripheral blood mononuclear cells (PBMC). To this end, we evaluated the gene expression profile of 19 genes (AKT2, CCND1, GSK3ALPHA, IGF1, GSK3BETA, FOXO3, IL6, IGFBP2, UGT2B17, ARA55, CREBBP, CYP11A, HSD17B1, HSD17B7, UGT2B7, SELADIN 1, CLU, PGC1, AKR1C1) selected according their function in the androgen pathways, in a series of 11 hypogonadal men pharmacologically treated with T. We noted that 7 genes were differentially expressed, five of them were up-regulated (AKT2 FC=2.39, CREBBP FC=11.2, GSK3beta FC=5.6, UGT2B7 FC=4.49, UGT2B17 FC=2.88) and two were down-regulated (ARA55 FC= -2.0, CYP11A FC= -2.47). This experience suggests that androgen- and insulin-related genes can be considered useful blood genomic biomarkers for specific steroid drugs.     

Fecal Calprotectin Concentrations in Healthy Children Aged 1-18 Months

Objective

Fecal calprotectin (FC) is an established biomarker of gut inflammation. The aim of this study was to evaluate FC concentrations in healthy children between 1 and 18 months of age.

Methods

Healthy children aged 1-18 months were enrolled in this study at the Department of Children''s Health Care in Shanghai, China. Children’s stool samples were collected and analyzed, and FC concentration was determined using a commercially available enzyme-linked immunosorbent assay (ELISA). The children''s weights and lengths were measured. Parents were asked to complete a brief questionnaire regarding several clinical and sociodemographic factors.

Results

The FC concentrations were unevenly distributed; the median FC concentration was 174.3 μg/g (range: 6.0-1097.7 μg/g) or 2.241 log10 μg/g (range: 0.775-3.041 log10 μg/g) for all 288 children. The children were divided into several age groups: 1-3 months, 3-6 months, 6-9 months, 9-12 months and 12-18 months. The median FC concentrations for these age groups were 375.2 μg/g (2.574 log10 μg/g), 217.9 μg/g (2.338 log10 μg/g), 127.7 μg/g (2.106 log10 μg/g), 96.1 μg/g (1.983 log10 μg/g) and 104.2 μg/g (2.016 log10 μg/g), respectively. A significant correlation between age and FC concentration was found (r=-0.490, p<0.001). A simple correlation analysis of weight-for-length Z-scores or weight-for-age Z-scores vs. FC concentrations showed that these variables were negatively correlated (Spearman’s rho=-0.287, p<0.001; Spearman’s rho=-0.243, p<0.001, respectively).

Conclusions

The FC levels of children aged 1-18 months exhibit a downward trend with increasing age and are greater than the normal levels observed in healthy adults. In healthy children aged <6 months, FC levels are high. In children aged 6-18 months, FC concentrations are relatively low but are still higher than those of children aged >4 years.     

Faecal levels of calprotectin in systemic sclerosis are stable over time and are higher compared to primary Sj?gren’s syndrome and rheumatoid arthritis

Introduction

Faecal calprotectin (FC) has been proposed to be a biomarker of gastrointestinal (GI) disease in systemic sclerosis (SSc). The purpose of this study was to extend cross-sectional observations and prospectively assess the variability of FC over time in SSc patients. We also aimed to examine FC in relation to immunosuppressive therapy. Finally we wanted to analyse FC in other rheumatic diseases to evaluate the specificity of FC for SSc GI disease.

Methods

FC was measured in consecutive patients with SSc, primary Sjögren’s syndrome (pSS), rheumatoid arthritis (RA) and in healthy hospital workers. The intraindividual variability of FC in SSc was assessed with intra class correlation (ICC) and κ statistics. Associations between FC and objective markers of GI disease and immunosuppressive medication were investigated.

Results

FC was associated with micronutrient deficiency and GI pathology as assessed by cineradiography confirming our previous results. FC showed only a limited intra-individual variation in SSc, ICC = 0.69 (95% confidence interval, CI: 0.57-0.78) and κ = 0.64 (95% CI: 0.56-0.73). Generalised immunosuppression did not have any significant impact on FC. FC was significantly higher in SSc patients compared to patients with pSS or RA as well as compared to healthy subjects.

Conclusions

FC is a promising non-invasive biomarker for GI disease in SSc. In view of stable levels over time, FC could be a useful marker when novel, more specific drugs targeting the GI tract in SSc will be introduced.     

Testing the role of apoA-I,HDL, and cholesterol efflux in the atheroprotective action of low-level apoE expression

Low levels of transgenic mouse apolipoprotein E (apoE) suppress atherosclerosis in apoE knockout (apoE-/-) mice without normalizing plasma cholesterol. To test whether this is due to facilitation of cholesterol efflux from the vessel wall, we produced apoA-I-/-/apoE-/- mice with or without the transgene. Even without apoA-I and HDL, apoA-I-/-/apoE-/- mice had the same amount of aorta cholesteryl ester as apoE-/- mice. Low apoE in the apoA-I-/-/apoE-/- transgenic mice reduced aortic lesions by 70% versus their apoA-I-/-/apoE-/- siblings. To define the free cholesterol (FC) efflux capacity of lipoproteins from the various genotypes, sera were assayed on macrophages expressing ATP-binding cassette transporter A1 (ABCA1). Surprisingly, ABCA1 FC efflux was twice as high to sera from the apoA-I-/-/apoE-/- or apoE-/- mice compared with wild-type mice, and this activity correlated with serum apoA-IV. Immunodepletion of apoA-IV from apoA-I-/-/apoE-/- serum abolished ABCA1 FC efflux, indicating that apoAI-V serves as a potent acceptor for FC efflux via ABCA1. With increasing apoE expression, apoA-IV and FC acceptor capacity decreased, indicating a reciprocal relationship between plasma apoE and apoA-IV. Low plasma apoE (1-3 x 10(-8) M) suppresses atherosclerosis by as yet undefined mechanisms, not dependent on the presence of apoA-I or HDL or an increased capacity of serum acceptors for FC efflux.