Endolysin LysEF-P10 shows potential as an alternative treatment strategy for multidrug-resistant Enterococcus faecalis infections.

Phage-derived lysins can hydrolyse bacterial cell walls and show great potential for combating Gram-positive pathogens. In this study, the potential of LysEF-P10, a new lysin derived from a isolated Enterococcus faecalis phage EF-P10, as an alternative treatment for multidrug-resistant E. faecalis infections, was studied. LysEF-P10 shares only 61% amino acid identity with its closest homologues. Four proteins were expressed: LysEF-P10, the cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain (LysEF-P10C), the putative binding domain (LysEF-P10B), and a fusion recombination protein (LysEF-P10B-green fluorescent protein). Only LysEF-P10 showed highly efficient, broad-spectrum bactericidal activity against E. faecalis. Several key functional residues, including the Cys-His-Asn triplet and the calcium-binding site, were confirmed using 3D structure prediction, BLAST and mutation analys. We also found that calcium can switch LysEF-P10 between its active and inactive states and that LysEF-P10B is responsible for binding E. faecalis cells. A single administration of LysEF-P10 (5 µg) was sufficient to protect mice against lethal vancomycin-resistant Enterococcus faecalis (VREF) infection, and LysEF-P10-specific antibody did not affect its bactericidal activity or treatment effect. Moreover, LysEF-P10 reduced the number of Enterococcus colonies and alleviated the gut microbiota imbalance caused by VREF. These results indicate that LysEF-P10 might be an alternative treatment for multidrug-resistant E. faecalis infections.

The Bacteriophage EF-P29 Efficiently Protects against Lethal Vancomycin-Resistant Enterococcus faecalis and Alleviates Gut Microbiota Imbalance in a Murine Bacteremia Model.

Enterococcus faecalis is becoming an increasingly important opportunistic pathogen worldwide, especially because it can cause life-threatening nosocomial infections. Treating E. faecalis infections has become increasingly difficult because of the prevalence of multidrug-resistant E. faecalis strains. Because bacteriophages show specificity for their bacterial hosts, there has been a growth in interest in using phage therapies to combat the rising incidence of multidrug-resistant bacterial infections. In this study, we isolated a new lytic phage, EF-P29, which showed high efficiency and a broad host range against E. faecalis strains, including vancomycin-resistant strains. The EF-P29 genome contains 58,984 bp (39.97% G+C), including 101 open reading frames, and lacks known putative virulence factors, integration-related proteins or antibiotic resistance determinants. In murine experiments, the administration of a single intraperitoneal injection of EF-P29 (4 × 105 PFU) at 1 h after challenge was sufficient to protect all mice against bacteremia caused by infection with a vancomycin-resistant E. faecalis strain (2 × 109 CFU/mouse). E. faecalis colony counts were more quickly eliminated in the blood of EF-P29-protected mice than in unprotected mice. We also found that exogenous E. faecalis challenge resulted in enrichment of members of the genus Enterococcus (family Enterococcaceae) in the guts of the mice, suggesting that it can enter the gut and colonize there. The phage EF-P29 reduced the number of colonies of genus Enterococcus and alleviated the gut microbiota imbalance that was caused by E. faecalis challenge. These data indicate that the phage EF-P29 shows great potential as a therapeutic treatment for systemic VREF infection. Thus, phage therapies that are aimed at treating opportunistic pathogens are also feasible. The dose of phage should be controlled and used at the appropriate level to avoid causing imbalance in the gut microbiota.

LysGH15 kills Staphylococcus aureus without being affected by the humoral immune response or inducing inflammation.

The lysin LysGH15, derived from the staphylococcal phage GH15, exhibits a wide lytic spectrum and highly efficient lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we found that LysGH15 did not induce resistance in MRSA or methicillin-sensitive S. aureus (MSSA) strains after repeated treatment. Although LysGH15 triggered the generation of LysGH15-specific antibodies in mice, these antibodies did not block lytic activity in vitro (nor the binding capacity of LysGH15). More importantly, when the antibody titre was highest in mice immunized with LysGH15, a single intravenous injection of LysGH15 was sufficient to protect mice against lethal infection with MRSA. These results indicated that LysGH15-specific antibodies did not affect the killing efficiency of LysGH15 against MRSA in vitro or in vivo. LysGH15 also reduced pro-inflammatory cytokines in mice with lethal infections. Furthermore, a high-dose LysGH15 injection did not cause significant adverse effects or pathological changes in the main organs of treated animals. These results provide further evidence for the administration of LysGH15 as an alternative strategy for the treatment of infections caused by MRSA.

Therapeutic effect of Pseudomonas aeruginosa phage YH30 on mink hemorrhagic pneumonia.

Hemorrhagic pneumonia caused by Pseudomonas aeruginosa remains one of the most costly infectious diseases among farmed mink and commonly leads to large economic losses during mink production. The objective of this study was to investigate the potential of using phages as a therapy against hemorrhagic pneumonia in mink. A broad-host-range phage from the Podoviridae family, YH30, was isolated using the mink-originating P. aeruginosa (serotype G) D7 strain as a host. The genome of YH30 was 72,192bp (54.92% G+C), contained 86 open reading frames and lacked regions encoding known virulence factors, integration-related proteins or antibiotic resistance determinants. These characteristics make YH30 eligible for use in phage therapy. The results of a curative treatment experiment demonstrated that a single intranasal administration of YH30 was sufficient to cure hemorrhagic pneumonia in mink. The mean colony count of P. aeruginosa in the blood and lung of YH30-protected mink was less than 10(3) CFU/mL (g) within 24h of bacterial challenge and ultimately became undetectable, whereas that in unprotected mink reached more than 10(8) CFU/mL (g). Additionally, YH30 dramatically improved the pathological manifestations of lung injury in mink with hemorrhagic pneumonia. Our work demonstrates the potential of phages to treat P. aeruginosa-caused hemorrhagic pneumonia in mink.

Characterization of Enterococcus faecium bacteriophage IME-EFm5 and its endolysin LysEFm5.

Due to the worldwide prevalence of antibiotic resistant strains, phages therapy has been revitalized recently. In this study, an Enterococcus faecium phage named IME-EFm5 was isolated from hospital sewage. Whole genomic sequence analysis demonstrated that IME-EFm5 belong to the Siphoviridae family, and has a double-stranded genome of 42,265bp (with a 35.51% G+C content) which contains 70 putative coding sequences. LysEFm5, the endolysin of IME-EFm5, contains an amidase domain in its N-terminal and has a wider bactericidal spectrum than its parental phage IME-EFm5, including 7 strains of vancomycin-resistant E. faecium. The mutagenesis analysis revealed that the zinc ion binding residues (H27, H132, and C140), E90, and T138 are required for the catalysis of LysEFm5. However, the antibacterial activity of LysEFm5 is zinc ion independent, which is inconsistent with most of other amidase members. The phage lysin LysEFm5 might be an alternative treatment strategy for infections caused by multidrug-resistant E. faecium.

Identification and characterization of HolGH15: the holin of Staphylococcus aureus bacteriophage GH15.

Holins are phage-encoded hydrophobic membrane proteins that spontaneously and non-specifically accumulate and form lesions in the cytoplasmic membrane. The ORF72 gene (also designated HolGH15) derived from the genome of the Staphylococcus aureus phage GH15 was predicted to encode a membrane protein. An analysis indicated that the protein encoded by HolGH15 potentially consisted of two hydrophobic transmembrane helices. This protein exhibited the structural characteristics of class II holins and belonged to the phage_holin_1 superfamily. Expression of HolGH15 in Escherichia coli BL21 cells resulted in growth retardation of the host cells, which was triggered prematurely by the addition of 2,4-dinitrophenol. The expression of HolGH15 caused morphological alterations in engineered E. coli cells, including loss of the cell wall and cytoplasmic membrane integrity and release of intracellular components, which were visualized by transmission electron microscopy. HolGH15 exerted efficient antibacterial activity at 37 °C and pH 5.2. Mutation analysis indicated that the two transmembrane domains of HolGH15 were indispensable for the activity of the full-length protein. HolGH15 showed a broad antibacterial range: it not only inhibited Staphylococcus aureus, but also demonstrated antibacterial activity against other species, including Listeria monocytogenes, Bacillus subtilis, Pseudomonas aeruginosa, Klebsiella pneumoniae and E. coli. At the minimal inhibitory concentration, HolGH15 evoked the release of cellular contents and resulted in the shrinkage and death of Staphylococcus aureus and Listeria monocytogenes cells. To the best of our knowledge, this study is the first report of a Staphylococcus aureus phage holin that exerts antibacterial activity against heterogeneous pathogens.

Combination Therapy of LysGH15 and Apigenin as a New Strategy for Treating Pneumonia Caused by Staphylococcus aureus.

Pneumonia is one of the most prevalent Staphylococcus aureus-mediated diseases, and the treatment of this infection is becoming challenging due to the emergence of multidrug-resistant S. aureus, especially methicillin-resistant S. aureus (MRSA) strains. It has been reported that LysGH15, the lysin derived from phage GH15, displays high efficiency and a broad lytic spectrum against MRSA and that apigenin can markedly diminish the alpha-hemolysin of S. aureus. In this study, the combination therapy of LysGH15 and apigenin was evaluated in vitro and in a mouse S. aureus pneumonia model. No mutual adverse influence was detected between LysGH15 and apigenin in vitro. In animal experiments, the combination therapy showed a more effective treatment effect than LysGH15 or apigenin monotherapy (P < 0.05). The bacterial load in the lungs of mice administered the combination therapy was 1.5 log units within 24 h after challenge, whereas the loads in unprotected mice or mice treated with apigenin or LysGH15 alone were 10.2, 4.7, and 2.6 log units, respectively. The combination therapy group showed the best health status, the lowest ratio of wet tissue to dry tissue of the lungs, the smallest amount of total protein and cells in the lung, the fewest pathological manifestations, and the lowest cytokine level compared with the other groups (P < 0.05). With regard to its better protective efficacy, the combination therapy of LysGH15 and apigenin exhibits therapeutic potential for treating pneumonia caused by MRSA. This paper reports the combination therapy of lysin and natural products derived from traditional Chinese medicine.

Therapeutic effect of the YH6 phage in a murine hemorrhagic pneumonia model.

The treatment, in farmed mink, of hemorrhagic pneumonia caused by multidrug-resistant Pseudomonas aeruginosa strains has become increasingly difficult. This study investigated the potential use of phages as a therapy against hemorrhagic pneumonia caused by P. aeruginosa in a murine hemorrhagic pneumonia model. An N4-like phage designated YH6 was isolated using P. aeruginosa strain D9. YH6 is a virulent phage with efficient and broad host lytic activity against P. aeruginosa. No bacterial virulence- or lysogenesis-related ORF is present in the YH6 genome, making it eligible for use in phage therapy. In our murine experiments, a single intranasal administration of YH6 (2 × 10(7) PFU) 2 h after D9 intranasal injections at double minimum lethal dose was sufficient to protect mice against hemorrhagic pneumonia. The bacterial load in the lungs of YH6-protected mice was less than 10(3) CFU/g within 24 h after challenge and ultimately became undetectable, whereas the amount of bacteria in the lung tissue derived from unprotected mice was more than 10(8) CFU/g within 24 h after challenge. In view of its protective efficacy in this murine hemorrhagic pneumonia model, YH6 may serve as an alternative treatment strategy for infections caused by multidrug-resistant P. aeruginosa.

A regulatory gene induces trichome formation and embryo lethality in tomato.

Trichomes are universal biological structures originating from the aerial epidermis, which serve as an excellent model to study plant differentiation at the cell level. Although the pathway regulating trichome formation in the Rosids has been well characterized, only very recently a few genes were identified for trichome initiation in the Asterids. In this study, we cloned Woolly (Wo), essential for trichome formation in tomato. Transgenic experiments revealed that the woolly phenotype is caused by the mutation in Wo which encodes a homeodomain protein containing a bZIP motif and a START domain. We identified three alleles of Wo and found that each allele contains a missense mutation, which respectively results in an amino acid substitution at the C terminus. Microarray and expression analysis showed that the expression of a B-type cyclin gene, SlCycB2, is possibly regulated by Wo, which also participates in trichome formation. Suppression of Wo or SlCycB2 expression by RNAi decreased the number of type I trichomes, and direct protein-protein interaction was detected between them, implying that both proteins may work together in the regulation of this type of trichome formation. Cytological observation and Wo transcript analysis in the developing seeds showed that embryo development was also correlated with Wo.

A multiple stress-responsive gene ERD15 from Solanum pennellii confers stress tolerance in tobacco.

Wild species often show more tolerance to environmental stress factors than their cultivated counterparts. An early responsive-to-dehydration gene was cloned from a drought- and salt-tolerant wild tomato Solanum pennellii (SpERD15). SpERD15 transcript accumulated differentially in different organs, and was remarkably induced by dehydration, salinity, cold and treatment with plant growth regulators. The protein encoded by SpERD15 was predominantly localized in the nucleus. Interestingly, we found that the majority of the transgenic tobacco plants were co-suppressed along with the overexpressing line. Overexpressing plants manifested stress tolerance accompanied by the accumulation of more soluble sugars and proline, and limited lipid peroxidation compared with co-suppression lines, which were more sensitive than the wild type. The differential contents of these compatible solutes in different transgenic lines were related to the changes in the expression of the genes involved in the production of some important osmolytes (P5CS and Sucrose synthase). Reduced lipid peroxidation over a broad range of stress factors was in agreement with increased expression of stress-responsive genes (ADH and GAPDH). Overexpression of SpERD15 increased the efficiency of PSII (F(v)/F(m)) in transgenic tobacco plants by maintaining PSII quinone acceptors in a partially oxidized form. The results show that SpERD15 augments stress tolerance by enhancing the efficiency of PSII through the protection of cellular membranes, as conferred by the accumulation of compatible solutes and limited lipid peroxidation.

Expression of artificial microRNAs in tomato confers efficient and stable virus resistance in a cell-autonomous manner.

Expression of artificial microRNAs (amiRNAs) in plants can target and degrade the invading viral RNA, consequently conferring virus resistance. Two amiRNAs, targeting the coding sequence shared by the 2a and 2b genes and the highly conserved 3' untranslated region (UTR) of Cucumber mosaic virus (CMV), respectively, were generated and introduced into the susceptible tomato. The transgenic tomato plants expressing amiRNAs displayed effective resistance to CMV infection and CMV mixed with non-targeted viruses, including tobacco mosaic virus and tomato yellow leaf curl virus. A series of grafting assays indicate scions originated from the transgenic tomato plant maintain stable resistance to CMV infection after grafted onto a CMV-infected rootstock. However, the grafting assay also suggests that the amiRNA-mediated resistance acts in a cell-autonomous manner and the amiRNA signal cannot be transmitted over long distances through the vascular system. Moreover, transgenic plants expressing amiRNA targeting the 2a and 2b viral genes displayed slightly more effective to repress CMV RNA accumulation than transgenic plants expressing amiRNA targeting the 3' UTR of viral genome did. Our work provides new evidence of the use of amiRNAs as an effective approach to engineer viral resistance in the tomato and possibly in other crops.

Overexpression of SlGMEs leads to ascorbate accumulation with enhanced oxidative stress, cold, and salt tolerance in tomato.

GDP-Mannose 3',5'-epimerase (GME; EC 5.1.3.18) catalyses the conversion of GDP-D-mannose to GDP-L-galactose, an important step in the ascorbic acid (AsA) biosynthesis pathway in higher plants. In this study, two members of the GME gene family were isolated from tomato (Solanum lycopersicum). Both SlGME genes encode 376 amino acids and share a 92% similarity with each other. Semi-quantitative RT-PCR indicated that SlGME1 was constantly expressed in various tissues, whereas SlGME2 was differentially expressed in different tissues. Transient expression of fused SlGME1-GFP (green fluorescent protein) and SlGME2-GFP in onion cells revealed the cytoplasmic localisation of the two proteins. Transgenic plants over-expressing SlGME1 and SlGME2 exhibited a significant increase in total ascorbic acid in leaves and red fruits compared with wild-type plants. They also showed enhanced stress tolerance based on less chlorophyll content loss and membrane-lipid peroxidation under methyl viologen (paraquat) stress, higher survival rate under cold stress, and significantly higher seed germination rate, fresh weight, and root length under salt stress. The present study demonstrates that the overexpression of two members of the GME gene family resulted in increased ascorbate accumulation in tomato and improved tolerance to abiotic stresses.

Over-expression of microRNA169 confers enhanced drought tolerance to tomato.

Plant miRNA regulates multiple developmental and physiological processes, including drought responses. We found that the accumulation of Sly-miR169 in tomato (Solanum lycopersicum) was induced by drought stress. Consequently, Sly-miR169 targets, namely, three nuclear factor Y subunit genes (SlNF-YA1/2/3) and one multidrug resistance-associated protein gene (SlMRP1), were significantly down-regulated by drought stress. Constitutive over-expression of a miR169 family member, Sly-miR169c, in tomato plant can efficiently down-regulate the transcripts of the target genes. Compared with non-transgenic plants, transgenic plants over-expressing Sly-miR169c displayed reduced stomatal opening, decreased transpiration rate, lowered leaf water loss, and enhanced drought tolerance. Our study is the first to provide evidence that the Sly-miR169c negatively regulates stomatal movement in tomato drought responses.

Transcriptional profiles of drought-responsive genes in modulating transcription signal transduction, and biochemical pathways in tomato.

To unravel the molecular mechanisms of drought responses in tomato, gene expression profiles of two drought-tolerant lines identified from a population of Solanum pennellii introgression lines, and the recurrent parent S. lycopersicum cv. M82, a drought-sensitive cultivar, were investigated under drought stress using tomato microarrays. Around 400 genes identified were responsive to drought stress only in the drought-tolerant lines. These changes in genes expression are most likely caused by the two inserted chromosome segments of S. pennellii, which possibly contain drought-tolerance quantitative trait loci (QTLs). Among these genes are a number of transcription factors and signalling proteins which could be global regulators involved in the tomato responses to drought stress. Genes involved in organism growth and development processes were also specifically regulated by drought stress, including those controlling cell wall structure, wax biosynthesis, and plant height. Moreover, key enzymes in the pathways of gluconeogenesis (fructose-bisphosphate aldolase), purine and pyrimidine nucleotide biosynthesis (adenylate kinase), tryptophan degradation (aldehyde oxidase), starch degradation (beta-amylase), methionine biosynthesis (cystathionine beta-lyase), and the removal of superoxide radicals (catalase) were also specifically affected by drought stress. These results indicated that tomato plants could adapt to water-deficit conditions through decreasing energy dissipation, increasing ATP energy provision, and reducing oxidative damage. The drought-responsive genes identified in this study could provide further information for understanding the mechanisms of drought tolerance in tomato.