Surface Display of Heterologous ß-Galactosidase in Food-Grade Recombinant Lactococcus lactis.

ß-Galactosidase is an essential enzyme for the metabolism of lactose in human beings and has an important role in the treatment of lactose intolerance (LI). ß-Galactosidase expressed by intestinal microflora, such as lactic acid bacteria (LAB), also alleviates LI. A promising approach to LI management is to exploit a food-grade LAB delivery system that can inhabit the human intestine and overproduce ß-galactosidase. In this study, we constructed a food-grade ß-galactosidase surface display delivery system and then integrated into the chromosome of Lactococcus lactis (L. lactis) NZ9000 using recombination. Western blot and immunofluorescence analyses confirmed that ß-galactosidase was expressed on the cell surface of recombinant L. lactis stain NZ-SDL. The whole-cell biocatalyst exhibits Vmax and Km values of 121.38 ± 7.17 UONPG/g and 65.36 ± 5.54 mM, based on ONPG hydrolysis. The optimum temperature for enzyme activity is 37 °C and the optimum pH is 5.0. Activity of the whole-cell biocatalyst is promoted by Mg2+, Ca2+, and K+, but inhibited by Zn2+, Fe2+, and Fe3+. The system has a thermal stability similar to purified ß-galactosidase but better pH stability, and is also more stable in artificial intestinal juice. Oral administration and intraperitoneal injections of NZ-SDL in mice cause no detectable health effects. In conclusion, we have successfully constructed a food-grade gene expression system in L. lactis that displays ß-galactosidase on the cell surface. This system exhibits good enzyme activity and stability in vitro, and is safe in vivo. It is therefore a promising candidate for use in LI management.

The chromosomal SezAT toxin-antitoxin system promotes the maintenance of the SsPI-1 pathogenicity island in epidemic Streptococcus suis.

Streptococcus suis has emerged as a causative agent of human meningitis and streptococcal toxic shock syndrome over the last years. The high pathogenicity of S. suis may be due in part to a laterally acquired pathogenicity island (renamed SsPI-1), which can spontaneously excise and transfer to recipients. Cells harboring excised SsPI-1 can potentially lose this island if cell division occurs prior to its reintegration; however, attempts to cure SsPI-1 from the host cells have been unsuccessful. Here, we report that an SsPI-1-borne Epsilon/Zeta toxin-antitoxin system (designated SezAT) promotes SsPI-1 stability in bacterial populations. The sezAT locus consists of two closely linked sezT and sezA genes encoding a toxin and its cognate antitoxin, respectively. Overproduction of SezT induces a bactericidal effect that can be neutralized by co-expression of SezA, but not by its later action. When devoid of a functional SezAT system, large-scale deletion of SsPI-1 is straightforward. Thus, SezAT serves to ensure inheritance of SsPI-1 during cell division, which may explain the persistence of epidemic S. suis. This report presents the first functional characterization of TA loci in S. suis, and the first biochemical evidence for the adaptive significance of the Epsilon/Zeta system in the evolution of pathogen virulence.

Unlocking the mystery of the hard-to-sequence phage genome: PaP1 methylome and bacterial immunity.

BACKGROUND: Whole-genome sequencing is an important method to understand the genetic information, gene function, biological characteristics and survival mechanisms of organisms. Sequencing large genomes is very simple at present. However, we encountered a hard-to-sequence genome of Pseudomonas aeruginosa phage PaP1. Shotgun sequencing method failed to complete the sequence of this genome. RESULTS: After persevering for 10 years and going over three generations of sequencing techniques, we successfully completed the sequence of the PaP1 genome with a length of 91,715 bp. Single-molecule real-time sequencing results revealed that this genome contains 51 N-6-methyladenines and 152 N-4-methylcytosines. Three significant modified sequence motifs were predicted, but not all of the sites found in the genome were methylated in these motifs. Further investigations revealed a novel immune mechanism of bacteria, in which host bacteria can recognise and repel modified bases containing inserts in a large scale. This mechanism could be accounted for the failure of the shotgun method in PaP1 genome sequencing. This problem was resolved using the nfi- mutant of Escherichia coli DH5α as a host bacterium to construct a shotgun library. CONCLUSIONS: This work provided insights into the hard-to-sequence phage PaP1 genome and discovered a new mechanism of bacterial immunity. The methylome of phage PaP1 is responsible for the failure of shotgun sequencing and for bacterial immunity mediated by enzyme Endo V activity; this methylome also provides a valuable resource for future studies on PaP1 genome replication and modification, as well as on gene regulation and host interaction.

A functional peptidoglycan hydrolase characterized from T4SS in 89K pathogenicity island of epidemic Streptococcus suis serotype 2.

BACKGROUND: Streptococcus suis serotype 2 (S. suis 2) has evolved efficient mechanisms to cause streptococcal toxic shock syndrome (STSS), which is a new emerging infectious disease linked to S. suis. We have previously reported that a type IV secretion system (T4SS) harbored by the specific 89K pathogenicity island (PAI) of S. suis 2 contributes to the development of STSS and mediates horizontal transfer of 89K. However, the 89K T4SS machinery assembly in vivo and in vitro is poorly understood, and the component acting directly to digest the bacterial cell wall needs to be identified. RESULTS: The virB1-89K gene product encoded in the 89K PAI is the only one that shows similarity to the Agrobacterium VirB1 component and contains a conserved CHAP domain that may function in peptidoglycan hydrolysis, which makes it a plausible candidate acting as a hydrolase against the peptidoglycan cell wall to allow the assembly of the T4SS apparatus. In the current study, the CHAP domain of VirB1-89K from S. suis 89K PAI was cloned and over-expressed in Escherichia coli, and its peptidoglycan-degrading activity in vitro was determined. The results indicated that the VirB1-89K CHAP domain can degrade the peptidoglycan layer of bacteria. Deletion of virB1-89K reduces significantly, but does not abolish, the virulence of S. suis in a mouse model. CONCLUSIONS: The experimental results presented here suggested that VirB1-89K facilitates the assembly of 89K T4SS apparatus by catalyzing the degradation of the peptidoglycan cell wall, thus contributing to the pathogenesis of S. suis 2 infection.

Use of bisphosphonates and the risk of osteonecrosis among cancer patients: a systemic review and meta-analysis of the observational studies.

PURPOSE: This study aims to systematically review observational studies evaluating the use of bisphosphonates (BPs) and risk of osteonecrosis of the jaw (ONJ) among cancer patients. METHODS: PubMed, Embase, and Cochrane Library were screened from database inception to Aug 2012. Two reviewers independently identified cohort and case-control studies evaluating the use of oral or intravenous (IV) BPs and the risk of ONJ and extracted the characteristics of the studies and risk estimates. Pooled estimates of odds ratios and 95 % confidence intervals were derived by random effects meta-analysis. Subgroup analyses were carried out according to patients' characteristics and route of BP use. RESULTS: We identified eight studies, including 1,389 cases and 569,620 controls. Use of BPs was associated with a significantly increased risk of ONJ (odds ratio (OR) 4.25; 95 % confidence interval (CI) 3.67-5.36; I (2) = 0 %). The summary OR was 4.22 (95 % CI 3.21-5.54; I (2) = 0 %) for adjusted studies. IV BPs were associated with higher risk (OR 4.27; 95 % CI 3.38-5.40; I (2) = 0 %) than oral BPs (OR 1.18; 95 % CI 0.89-1.56; I (2) = 0 %). Hospital-based studies were associated with higher risk estimates than population-based studies. CONCLUSION: The available evidence suggests that use of BPs in cancer patients is associated with a substantial risk for ONJ. Patients receiving IV BP are at the highest risk. It is important to assess oral health before initiating therapy and to avoid dental procedures during the active phase of intravenous BP therapy.

Characterization and genome sequencing of phage Abp1, a new phiKMV-like virus infecting multidrug-resistant Acinetobacter baumannii.

While screening for alternative antibiotics against multidrug-resistant Acinetobacter baumannii, we isolated a virulent A. baumannii bacteriophage Abp1. Transmission electron microscopy revealed that the phage had an icosahedral head with a short tail and should be classified as a member of the Podoviridae family. SDS-PAGE showed that Abp1 contained at least one major and nine minor proteins. In a single-step growth test, we demonstrated that Abp1 had a latent period of 10 min and a burst size of 350. Abp1 also had a relatively narrow host range. The entire genome was sequenced, and the final assembly yielded a 42,185 bp, linear, double-stranded DNA molecule with a G+C content of 39.15 % and containing 54 putative genes. Among these genes, 26 were functionally known, leaving 28 unknown putative genes. Abp1 is a new member of the phiKMV-like virus subgroup of the T7 group; its genome sequence is very similar to that of the A. baumannii phage phiAB1.

[Construction and virulence evaluation of the virB1-89K gene knockout mutant of type IV-like secretion system of Streptococcus suis serotype 2].

OBJECTIVE: To construct the virB1-89K gene knockout mutant and its complementary strain of Streptococcus suis serotype 2 (SS2) highly virulent strain 05ZYH33 and evaluate the role of virB1-89K in the pathogenesis of SS2. METHODS: The virB1-89K gene was knocked out by homologous recombination, then multiple-PCR and sequence analysis were used to identify the knockout strain deltavirB1-89K. The virB1-89K gene and its upstream promoter were cloned into the E. coli-S. suis shuttle vector pSET1, and the recombinant plasmid was electrotransformed into the deltavirB1-89K mutant to generate the complementary strain CvirB1-89K. The effects of virB1-89K deletion on the basic biological characteristics and virulence of SS2 were then determined in this study. RESULTS: The isogenic mutant deltavirB1-89K and its complementary strain CvirB1-89K were successfully constructed. No significant differences in biological characteristics were found among the three strains. However, the virulence of the deltavirB1-89K mutant was reduced to 30% of the wild-type level and functional complementation of virB1-89K restored its pathogenicity. CONCLUSION: The virB1-89K gene plays an important role in the pathogenesis of S. suis 2 infection.

Comparative genomics and DNA methylation analysis of Pseudomonas aeruginosa clinical isolate PA3 by single-molecule real-time sequencing reveals new targets for antimicrobials.

Introduction: Pseudomonas aeruginosa (P.aeruginosa) is an important opportunistic pathogen with broad environmental adaptability and complex drug resistance. Single-molecule real-time (SMRT) sequencing technique has longer read-length sequences, more accuracy, and the ability to identify epigenetic DNA alterations. Methods: This study applied SMRT technology to sequence a clinical strain P. aeruginosa PA3 to obtain its genome sequence and methylation modification information. Genomic, comparative, pan-genomic, and epigenetic analyses of PA3 were conducted. Results: General genome annotations of PA3 were discovered, as well as information about virulence factors, regulatory proteins (RPs), secreted proteins, type II toxin-antitoxin (TA) pairs, and genomic islands. A genome-wide comparison revealed that PA3 was comparable to other P. aeruginosa strains in terms of identity, but varied in areas of horizontal gene transfer (HGT). Phylogenetic analysis showed that PA3 was closely related to P. aeruginosa 60503 and P. aeruginosa 8380. P. aeruginosa's pan-genome consists of a core genome of roughly 4,300 genes and an accessory genome of at least 5,500 genes. The results of the epigenetic analysis identified one main methylation sites, N6-methyladenosine (m6A) and 1 motif (CATNNNNNNNTCCT/AGGANNNNNNNATG). 16 meaningful methylated sites were picked. Among these, purH, phaZ, and lexA are of great significance playing an important role in the drug resistance and biological environment adaptability of PA3, and the targeting of these genes may benefit further antibacterial studies. Disucssion: This study provided a detailed visualization and DNA methylation information of the PA3 genome and set a foundation for subsequent research into the molecular mechanism of DNA methyltransferase-controlled P. aeruginosa pathogenicity.

Functional identification of the DNA packaging terminase from Pseudomonas aeruginosa phage PaP3.

Terminase proteins are responsible for DNA recognition and initiation of DNA packaging in phages. We previously reported the genomic sequence of a temperate Pseudomonas aeruginosa phage, PaP3, and determined its precise integration site in the host bacterial chromosome. In this study, we present a detailed functional identification of the DNA packaging terminase for phage PaP3. The purified large subunit p03 was demonstrated to possess ATPase and nuclease activities, as well as the ability to bind to specific DNA when it is unassembled. In addition, a small terminase subunit (p01) of a new type was found and shown to bind specifically to cos-containing DNA and stimulate the cos-cleavage and ATPase activities of p03. The results presented here suggest that PaP3 utilizes a typical cos site mechanism for DNA packaging and provide a first step towards understanding the molecular mechanism of the PaP3 DNA packaging reaction.

Eliciting cross-neutralizing antibodies in mice challenged with a dengue virus envelope domain III expressed in Escherichia coli.

Dengue viruses (DENVs) are mosquito-borne infectious pathogens that pose a serious global public health threat, and at present, no therapy or effective vaccines are available. Choosing suitable units as candidates is fundamental for the development of a dengue subunit vaccine. Domain III of the DENV-2 E protein (EDIII) was chosen in the present study and expressed in Escherichia coli by N-terminal fusion to a bacterial leader (pelB), and C-terminal fusion with a 6×His tag based on the functions of DENV structure proteins, especially the neutralizing epitopes on the envelope E protein. After two-step purification using Ni-NTA affinity and cation-exchange chromatography, the His-tagged EDIII was purified up to 98% homogenicity. This recombinant EDIII was able to trigger high levels of neutralizing antibodies in both BALB/c and C57BL/6 mice. Both the recombinant EDIII and its murine antibodies protected Vero cells from DENV-2 infection. Interestingly, the recombinant EDIII provides at least partial cross-protection against DENV-1 infection. In addition, the EDIII antibodies were able to protect suckling mice from virus challenge in vivo. These data suggest that a candidate molecule based on the small EDIII protein, which has neutralizing epitopes conserved among all 4 DENV serotypes, has important implications.

Long noncoding RNA ADIRF antisense RNA 1 upregulates insulin receptor substrate 1 to decrease the aggressiveness of osteosarcoma by sponging microRNA-761.

An increasing number of studies have supported the critical regulatory actions of long noncoding RNAs (lncRNAs) in osteosarcoma (OS). However, the detailed roles of adipogenesis regulatory factor-antisense RNA 1 (ADIRF-AS1) in OS have not been comprehensively described. Hence, we first detected ADIRF-AS1 expression in OS and evaluated its clinical significance. Functional experiments were then performed to determine the modulatory role of ADIRF-AS1 in OS progression. ADIRF-AS1 was found to be overexpressed in OS, and the overall survival of patients with OS who had high ADIRF-AS1 levels was shorter than that of those with low levels. ADIRF-AS1 knockdown led to restricted proliferation, migration, and invasiveness of OS cells and increased apoptosis. Additionally, ADIRF-AS1 downregulation impeded tumor growth in vivo. Mechanistically, ADIRF-AS1 acted as a competitive endogenous RNA for microRNA-761 (miR-761) that siphoned miR-761 away from its target, namely insulin receptor substrate 1 (IRS1), leading to IRS1 overexpression. Rescue experiments showed that low levels of miR-761 or restoration of IRS1 could neutralize the effects of ADIRF-AS1 ablation in OS cells. In summary, ADIRF-AS1 exacerbates the oncogenicity of the OS cells by targeting the miR-761/IRS1 axis. Our findings may aid in the advancement of lncRNA-directed therapeutics for OS.

Long non­coding RNA LINC01748 exerts carcinogenic effects in non­small cell lung cancer cell lines by regulating the microRNA­520a­5p/HMGA1 axis.

The important functions of long non­coding RNAs in the malignancy of non­small cell lung cancer (NSCLC) has been increasingly highlighted. However, whether LINC01748 functions in a crucial regulatory role still requires further research. The aim of the present study was to investigate the biological roles of LINC01748 in NSCLC. Furthermore, different experiments were utilized to investigate the mechanism of action of LINC01748 in 2 NSCLC cell lines. Reverse transcription­quantitative PCR was used to measure mRNA expression levels. Cell Counting Kit­8 assay, flow cytometry analysis and Transwell and Matrigel assays were also used to analyze, cell viability, apoptosis, and migration and invasion, respectively. A tumor xenograft model was used for in vivo experiments. RNA immunoprecipitation experiments, luciferase reporter assays and rescue experiments were used to investigate the mechanisms involved. Data from The Cancer Genome Atlas dataset and patients recruited into the present study showed that LINC01748 was overexpressed in NSCLC. Patients with high LINC01748 mRNA expression level had shorter overall survival rate compared with that in patients with low LINC01748 mRNA expression level. Then, knockdown of LINC01748 mRNA expression level reduced cell proliferation, migration and invasion, but increased cell apoptosis in vitro. Knockdown of LINC01748 also reduced tumor growth in vivo. Mechanistically, LINC01748 could act as a competing endogenous (ce)RNA to sponge microRNA(miR)­520a­5p, to increase the expression level of the target gene, high mobility group AT­hook 1 (HMGA1) in the NSCLC cell lines. Furthermore, rescue experiments illustrated that the functions exerted by LINC01748 knockdown were negated by miR­520a­5p inhibition or HMGA1 overexpression. In summary, LINC01748 acted as a ceRNA by sponging miR­520a­5p, leading to HMGA1 overexpression, thus increasing the aggressiveness of the NSCLC cells. Accordingly, targeting the LINC01748/miR­520a­5p/HMGA1 pathway may benefit NSCLC therapy.

Specific Premature Groups Have Better Benefits When Treating Apnea With Caffeine Than Aminophylline/Theophylline.

Background: Methylxanthines (caffeine; aminophylline/theophylline) are commonly used for apnea of prematurity (AOP) treatment. We aimed to compare the efficacy and adverse effects of caffeine and aminophylline/theophylline. Methods: A retrospective case-control gestational age-matched study investigates patients born between January 2017 and December 2018, 23-35 weeks gestation with birth weights >500 g treating AOP with caffeine or aminophylline/theophylline. Results: There were 144 cases (48 in caffeine group and 96 in aminophylline/theophylline group). The median treatment durations were 11 and 17 days in caffeine and aminophylline/theophyllinegroup (p = 0.002). When tachycardia is defined as heart rate ≥160 bpm, the rates were 8.3 and 34.4% in caffeine and control group (p = 0.001). When tachycardia is defined as 10 bpm over baseline heart rate, the rates were 41.7 and 63.5% in caffeine and aminophylline/theophylline group (p = 0.01). Stratified by gestational age and sex, significant reductions in tachycardia rates with caffeine than with theophylline were limited to male infants and infants born at <30 weeks gestation. Conclusions: For apnea treatment, caffeine has greater efficacy and fewer tachycardia than aminophylline/theophylline, especially in male infants and infants born at <30 weeks gestation.

Recombinant antimicrobial peptide hPAB-ß expressed in Pichia pastoris, a potential agent active against methicillin-resistant Staphylococcus aureus.

As a potential therapeutic agent, antimicrobial peptide has received increased attention in recent years. However, high-level expression of a small peptide with antimicrobial activity is still a challenging task. In this study, the coding sequence of antimicrobial peptide hPAB-ß, a variant derived from human beta-defensin 2, was cloned into pPIC9K vector and transformed into Pichia pastoris. P. pastoris transformants harbored with multi-copy plasmids were screened by G418 selection. When the transformed cells were induced by methanol, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, and matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed recombinant hPAB-ß products consisting of three protein species of 4,680.4, 4,485.3, and 4,881.9 Da at proportions of 58%, 36%, and 6%, respectively, which may be due to the incomplete processing of the fusion signal peptide of α-factor by the STE13 protease. Expressed hPAB-ß was secreted into the culture medium at a level of 241.2 ± 29.5 mg/L. Purified hPAB-ß with 95% homogeneity was obtained by 10 kDa membrane filtration followed by cation ion-exchange chromatography with a SP-Sepharose XL column. The two major protein species separated through a SOURCE 30RPC reverse phase chromatography column showed definite antimicrobial activities against Staphylococcus aureus. All 22 methicillin-resistant S. aureus (MRSA) isolates with multidrug resistance phenotype were sensitive to the recombinant hPAB-ß with minimal inhibitory concentrations of 8-64 µg/ml. Our results show that the methylotrophic yeast-inducible system is suitable for high-level expression of active hPAB-ß, and that expressed hPAB-ß in P. pastoris may be a potential antimicrobial agent against MRSA infection.

Identification of lytic bacteriophage MmP1, assigned to a new member of T7-like phages infecting Morganella morganii.

MmP1 (Morganella morganii phage 1) is a lytic bacteriophage newly isolated from the host bacterium M. morganii. The entire genome was sequenced, and final assembly yielded a 38,234bp linear double-stranded DNA (dsDNA) with a G+C content of 46.5%. In the MmP1 genome, 49 putative genes, 10 putative promoters and 2 predicted sigma-independent terminators were determined through bioinformatic analysis. A striking feature of the MmP1 genome is its high degree of similarity to the T7 group of phages. All of the 49 predicted genes exist on the same DNA strand, and functions were assigned to 35 genes based on the similarity of the homologues deposited in GenBank, which share 30-80% identity to their counterparts in T7-like phages. The analyses of MmP1 using CoreGenes, phylogenetic tree of RNA polymerase and structural proteins have demonstrated that bacteriophage MmP1 should be assigned as a new member of T7-like phages but as a relatively distant member of this family. This is the first report that a T7-like phage adaptively parasitizes in M. morganii, and this will advance our understanding of biodiversity and adaptive evolution of T7-like phages.

The Effect of Dark Septate Endophytic Fungi on Mahonia oiwakensis.

This is the first study to discuss the effects of dark septate endophytes (DSE) on the growth promotion and berberine concentration in Mahonia oiwakensis, whose extract (MOE) has been suggested to have potential therapeutic effects against human lung cancer. First, as per phylogenetic analysis, the strains were divided into four groups: CkDB2, CkDB5, MoAL2 and MoAL5. All of these were DSEs, which could form microsclerotia in M. oiwakensis. The growth response experiment revealed that inoculation of the plant with MoAL5 and CkDB5 promoted an increase in the total fresh weight of the seedlings. Chemical composition analysis showed that seedlings inoculated with CkDB5 had the highest berberine concentration. These results showed that some DSEs have the ability to promote growth and induce phytochemical responses in the host plant.

Hypophosphatemia as an Early Metabolic Bone Disease Marker in Extremely Low-Birth-Weight Infants After Prolonged Parenteral Nutrition Exposure.

BACKGROUND: Early metabolic bone disease (MBD) detection is important in preterm infants to decrease long-term consequence. We aim to explore the early MBD biochemical marker in extremely low-birth-weight (ELBW) infants. METHODS: Retrospective cohort study of 95 preterm infants born in a tertiary care-level neonatal intensive care unit between January 2015 and June 2018, with birth weight <1000 g. Thirty-five infants were "nothing by mouth" for >14 days and categorized as the high-risk group; the remaining 60 were categorized as the control group. Mineral intake in the first 14 days and the trend of serum calcium (Ca), phosphorus (P), and alkaline phosphatase (ALP) levels were compared in both groups. RESULTS: The Ca and P supplementation in the first 2 weeks of life were inadequate in both groups. Compared with the control group, significantly lower serum P (mg/dL) levels were noted in the high-risk group on weeks 2 (3.65 ± 1.2 vs 4.67 ± 1.45; P < .001), 4 (3.21 ± 0.95 vs 5.83 ± 1.18; P < .0001), and 6 (3.94 ± 1.1 vs 6.22 ± 0.78; P <.0001). There was no significant difference in the serum Ca level, and significantly higher ALP (U/L) levels were found up until 2 months of life in the high-risk group (458.36 ± 189.02 vs 335.7 ± 111.51; P < .014). CONCLUSION: Hypophosphatemia developed as early as 2 weeks old in high-risk preterm infants because of inadequate supplementation. Neither the serum Ca or ALP levels were affected. Thus, the routine monitoring of serum P level should be started 2 weeks after birth for early MBD detection in extremely ELBW infants.

Adaptation of Pseudomonas aeruginosa to Phage PaP1 Predation via O-Antigen Polymerase Mutation.

Adaptation of bacteria to phage predation poses a major obstacle for phage therapy. Bacteria adopt multiple mechanisms, such as inhibition of phage adsorption and CRISPR/Cas systems, to resist phage infection. Here, a phage-resistant mutant of Pseudomonas aeruginosa strain PA1 under the infection of lytic phage PaP1 was selected for further study. The PaP1-resistant variant, termed PA1RG, showed decreased adsorption to PaP1 and was devoid of long chain O-antigen on its cell envelope. Whole genome sequencing and comparative analysis revealed a single nucleotide mutation in the gene PA1S_08510, which encodes the O-antigen polymerase Wzy that is involved in lipopolysaccharide (LPS) biosynthesis. PA1_Wzy was classified into the O6 serotype based on sequence homology analysis and adopts a transmembrane topology similar to that seem with P. aeruginosa strain PAO1. Complementation of gene wzy in trans enabled the mutant PA1RG to produce the normal LPS pattern with long chain O-antigen and restored the susceptibility of PA1RG to phage PaP1 infection. While wzy mutation did not affect bacterial growth, mutant PA1RG exhibited decreased biofilm production, suggesting a fitness cost of PA1 associated with resistance of phage PaP1 predation. This study uncovered the mechanism responsible for PA1RG resistance to phage PaP1 via wzy mutation and revealed the role of phages in regulating bacterial behavior.

Antibacterial Activity of a Lytic Enzyme Encoded by Pseudomonas aeruginosa Double Stranded RNA Bacteriophage phiYY.

Multidrug-resistant Pseudomonas aeruginosa is one of the most life-threatening pathogens for global health. In this regard, phage encoded lytic proteins, including endolysins and virion-associated peptidoglycan hydrolases (VAPGH), have been proposed as promising antimicrobial agents to treat P. aeruginosa. Most dsDNA phages use VAPGH to degrade peptidoglycan (PG) during infection, and endolysin to lyse the host cells at the end of lytic cycle. By contrast, dsRNA phage encodes only one lytic protein, which is located in the viral membrane to digest the PG during penetration, and also serves as an endolysin to release the phage. Currently, there are only seven sequenced dsRNA phages, and phiYY is the only one that infects human pathogen P. aeruginosa. In this study, dsRNA phage phiYY encoded lysin, named Ply17, was cloned and purified. Ply17 contains a PG-binding domain and a lysozyme-like-family domain. Ply17 exhibited a broad antibacterial activity against the outer membrane permeabilizer treated Gram-negative bacteria. The best lytic activity was achieved at 37°C, pH 7.5, in the presence of 0.5 mM EDTA. Moreover, it could effectively lyse Gram-positive bacteria directly, including Staphylococcus aureus. Therefore, dsRNA phage encoded Ply17 might be a promising new agent for treating multidrug-resistant pathogens.

Gut Microbiota is Altered in Patients with Alzheimer's Disease.

Previous studies suggest that gut microbiota is associated with neuropsychiatric disorders, such as Parkinson's disease, amyotrophic lateral sclerosis, and depression. However, whether the composition and diversity of gut microbiota is altered in patients with Alzheimer's disease (AD) remains largely unknown. In the present study, we collected fecal samples from 43 AD patients and 43 age- and gender-matched cognitively normal controls. 16S ribosomal RNA sequencing technique was used to analyze the microbiota composition in feces. The composition of gut microbiota was different between the two groups. Several bacteria taxa in AD patients were different from those in controls at taxonomic levels, such as Bacteroides, Actinobacteria, Ruminococcus, Lachnospiraceae, and Selenomonadales. Our findings suggest that gut microbiota is altered in AD patients and may be involved in the pathogenesis of AD.