Guangtao Huang

Mapping the tail fiber as the receptor binding protein responsible for differential host specificity of Pseudomonas aeruginosa bacteriophages PaP1 and JG004.

The first step in bacteriophage infection is recognition and binding to the host receptor, which is mediated by the phage receptor binding protein (RBP). Different RBPs can lead to differential host specificity. In many bacteriophages, such as Escherichia coli and Lactococcal phages, RBPs have been identified as the tail fiber or protruding baseplate proteins. However, the tail fiber-dependent host specificity in Pseudomonas aeruginosa phages has not been well studied. This study aimed to identify and investigate the binding specificity of the RBP of P. aeruginosa phages PaP1 and JG004. These two phages share high DNA sequence homology but exhibit different host specificities. A spontaneous mutant phage was isolated and exhibited broader host range compared with the parental phage JG004. Sequencing of its putative tail fiber and baseplate region indicated a single point mutation in ORF84 (a putative tail fiber gene), which resulted in the replacement of a positively charged lysine (K) by an uncharged asparagine (N). We further demonstrated that the replacement of the tail fiber gene (ORF69) of PaP1 with the corresponding gene from phage JG004 resulted in a recombinant phage that displayed altered host specificity. Our study revealed the tail fiber-dependent host specificity in P. aeruginosa phages and provided an effective tool for its alteration. These contributions may have potential value in phage therapy.

Genomic and Proteomic Analyses of the Terminally Redundant Genome of the Pseudomonas aeruginosa Phage PaP1: Establishment of Genus PaP1-Like Phages

We isolated and characterized a new Pseudomonas aeruginosa myovirus named PaP1. The morphology of this phage was visualized by electron microscopy and its genome sequence and ends were determined. Finally, genomic and proteomic analyses were performed. PaP1 has an icosahedral head with an apex diameter of 68–70 nm and a contractile tail with a length of 138–140 nm. The PaP1 genome is a linear dsDNA molecule containing 91,715 base pairs (bp) with a G+C content of 49.36% and 12 tRNA genes. A strategy to identify the genome ends of PaP1 was designed. The genome has a 1190 bp terminal redundancy. PaP1 has 157 open reading frames (ORFs). Of these, 143 proteins are homologs of known proteins, but only 38 could be functionally identified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography-mass spectrometry allowed identification of 12 ORFs as structural protein coding genes within the PaP1 genome. Comparative genomic analysis indicated that the Pseudomonas aeruginosa phage PaP1, JG004, PAK_P1 and vB_PaeM_C2-10_Ab1 share great similarity. Besides their similar biological characteristics, the phages contain 123 core genes and have very close phylogenetic relationships, which distinguish them from other known phage genera. We therefore propose that these four phages be classified as PaP1-like phages, a new phage genus of Myoviridae that infects Pseudomonas aeruginosa.

Global Transcriptomic Analysis of Interactions between Pseudomonas aeruginosa and Bacteriophage PaP3.

The interactions between Bacteriophage (phage) and host bacteria are widespread in nature and influences of phage replication on the host cells are complex and extensive. Here, we investigate genome-wide interactions of Pseudomonas aeruginosa (P. aeruginosa) and its temperate phage PaP3 at five time points during phage infection. Compared to the uninfected host, 38% (2160/5633) genes of phage-infected host were identified as differentially expressed genes (DEGs). Functional analysis of the repressed DEGs revealed infection-stage-dependent pathway communications. Based on gene co-expression analysis, most PaP3 middle genes were predicted to have negative impact on host transcriptional regulators. Sub-network enrichment analysis revealed that adjacent genes of PaP3 interacted with the same host genes and might possess similar functions. Finally, our results suggested that during the whole infection stage, the early genes of PaP3 had stronger regulatory role in host gene expression than middle and late genes, while the host genes involved amino acid metabolism were the most “vulnerable” targets of these phage genes. This work provides the basis for understanding survival mechanisms of parasites and host, and seeking phage gene products that could potentially be used in anti-bacterial infection.

Antibacterial properties of Acinetobacter baumannii phage Abp1 endolysin (PlyAB1)

BackgroundAcinetobacter baumannii has emerged as one of the most important hospital-acquired pathogens in the world, because of its resistance to almost all available antibiotic drugs. Endolysins from phages are attracting increasing interest as potential antimicrobial agents, especially for drug-resistant bacteria. We previously isolated and characterized Abp1, a virulent phage targeting the multidrug-resistant A. baumannii strain, AB1.MethodsTo evaluate the antimicrobial potential of endolysin from the Abp1 phage, the endolysin gene plyAB1 was cloned and over-expressed in Escherichia coli, and the lytic activity of the recombinant protein (PlyAB1) was tested by turbidity assessment and bacteria counting assays.ResultsPlyAB1 exhibits a marked lytic activity against A. baumannii AB1, as shown by a decrease in the number of live bacteria following treatment with the enzyme. Moreover, PlyAB1 displayed a highly specific lytic effect against all of the 48 hospital-derived pandrug-resistant A. baumannii isolates that were tested. These isolates were shown to belong to different ST clones by multilocus sequence typing.ConclusionsThe results presented here show that PlyAB1 has potential as an antibiotic against drug-resistant A. baumannii.

Astragaloside-IV Alleviates Heat-Induced Inflammation by Inhibiting Endoplasmic Reticulum Stress and Autophagy

Background: Thermal injury is the main cause of pulmonary disease in stroke after burn and can be life threatening. Heat-induced inflammation is an important factor that triggers a series of induces pathological changes. However, this mechanism underlying heat-induced inflammation in thermal inhalation injury remains unclear. Studies have revealed that astragaloside-IV (AS-IV), a natural compound extracted from Astragalus membranaceus, has protective effects in inflammatory diseases. Here, we investigated whether the protective effects of AS-IV occur because of the suppression of heat-induced endoplasmic reticulum (ER) stress and excessive autophagy Methods: AS-IV was administered to Wistar rats after thermal inhalation injury and 16HBE140-cells were treated with AS-IV. TNF-α, IL-6, and IL-8 levels were determined by ELISA and real-time PCR. ER stress and autophagy were determined by western blot. Autophagic flux was measured by recording the fluorescence emission of the fusion protein mRFP-GFP-LC3 by dynamic live-cell imaging. Results: AS-IV had protective effects against heat-induced reactive oxygen species production and attenuated ER stress. AS IV alleviated heat-induced excessive autophagy in vitro and in vivo. Excessive autophagy was attenuated by the PERK inhibitor GSK2656157 and eIF2α siRNA, suggesting that heat stress-induced autophagy can activate the PERK-eIF2α pathway. Beclin 1 and Atg5 siRNAs inhibited the upregulation of the inflammatory cytokines TNF-α, IL-6, and IL-8 after heat exposure. Conclusions: Thus, AS-IV may attenuate inflammatory responses by disrupting the crosstalk between autophagy and the PERK-eIF2α pathway and may be an ideal agent for treating inflammatory pulmonary diseases.

Multilocus Sequence Typing Analysis of Carbapenem-Resistant Acinetobacter baumannii in a Chinese Burns Institute

Acinetobacter baumannii is a leading pathogen responsible for nosocomial infections. The emergence of carbapenem-resistant A. baumannii (CRAB) has left few effective antibiotics for clinicians to use. To investigate the temporal evolutionary relationships among CRAB strains, we collected 248 CRAB isolates from a Chinese burns institute over 3 years. The prevalence of the OXA-23 gene was detected by polymerase chain reaction. Multilocus sequence typing was used to type the CRAB strains and eBURST was used to analyze their evolutionary relationships. Wound surfaces (41%), sputa (24%), catheters (15%), and bloods (14%) were the four dominant isolation sources. Except for minocycline (33.5%) and sulbactam/cefoperazone (74.6%), these CRAB strains showed high resistance rates (>90%) to 16 tested antibiotics. The 248 isolates fall into 26 sequence types (STs), including nine known STs and 17 unknown STs. The majority (230/248) of these isolates belong to clonal complex 92 (CC92), including eight isolates belonging to seven unreported STs. A new CC containing 11 isolates grouped into four new STs was identified. The OXA-23 gene was detected at high prevalence among the CRAB isolates and the prevalence rate among the various STs differed. The majority of the isolates displayed a close evolutionary relationship, suggesting that serious nosocomial spreading and nosocomial infections of CRAB have occurred in the burn unit. In conclusion, the main CC for CRAB in this Chinese burn unit remained unchanged during the 3-year study period, and a new CC was identified. CC92 was the dominant complex, and more attention should be directed toward monitoring the new CC we have identified herein.

Burn Serum Increases Staphylococcus aureus Biofilm Formation via Oxidative Stress

Staphylococcus aureus is a common pathogen isolated from burn patients that can form biofilms on burn wounds and implanted deep vein catheters, which often leads to refractory infections or even biofilm-related sepsis. As biofilm formation is usually regulated by environmental conditions, we hypothesized that serum composition may be altered after burn injury, potentially affecting the ability of infecting bacteria to form biofilms. As predicted, we observed that serum from burn-injured rats increases biofilm formation by S. aureus and also induces bacterial aggregation and adherence to human fibronectin and fibrinogen. Analysis of potential regulatory factors revealed that exposure to burn serum decreases expression of the quorum-sensing agr system and increases mRNA levels of some biofilm inducers such as sarA and icaA. In addition, we also observed that burn serum imposes oxidative stress and increases expression of key oxidoreductase genes (sodA, sodM, katA, and ahpC) in S. aureus. Importantly, the ability of burn serum to enhance biofilm formation and bacterial cell aggregation can be abrogated by treatment with an antioxidant. Taken together, these findings indicate that burn serum increases S. aureus biofilm formation via elevated oxidative stress, and may lead to novel strategies to control biofilm formation and infection in burn patients.

A 5-year Survey Reveals Increased Susceptibility to Glycopeptides for Methicillin-Resistant Staphylococcus aureus Isolates from Staphylococcus aureus Bacteremia Patients in a Chinese Burn Center

Methicillin-resistant Staphylococcus aureus (MRSA) infections are prevalent in burn wards, and are especially serious in S. aureus bacteremia (SAB) patients. Glycopeptides and daptomycin are effective against MRSA infections, but MIC creeps can reduce their efficacy. Our object was to perform a molecular epidemiological investigation of S. aureus isolates in our burn center and to evaluate MICs for antimicrobials against SAB-associated MRSA isolates. A total of 259 S. aureus isolates, obtained from August 2011 to July 2016, were used in this study. Multiple molecular typing was used for molecular epidemiological analysis. E-tests were used to determine MICs of vancomycin, teicoplanin, and daptomycin for SAB-associated MRSA isolates. MIC values were stratified by collection date or source and compared. Spearmans test was used to analyze MICs correlations amongst tested antimicrobials. ST239-MRSA-III-t030-agrI clone was found to be dominant in both SAB and non-SAB patients, and significantly more in SAB patients (P < 0.0001). SAB-MRSA isolates exhibited decreased MICs for vancomycin, teicoplanin, and daptomycin during the 5-year period. Compared to those isolated from catheters or wounds, SAB-MRSA isolates from the bloodstream were less susceptible to vancomycin and daptomycin, but more susceptible to teicoplanin. MICs Correlation was found only between vancomycin and daptomycin in MRSA isolates from the bloodstream (rho = 0.250, P = 0.024). In conclusion, our results suggest that MRSA infections are still serious problems in burn centers. In contrast to most other studies, we observed increased susceptibility to glycopeptides and daptomycin against SAB-associated MRSA in our center from 2011 to 2016, suggesting the use of glycopeptides does not lead to MIC creeps. Isolates from different sites of the body may exhibit different levels of susceptibility and change trend over time for different antimicrobials, antimicrobials selection for MRSA infections should be considered comprehensively.

Epidemiology and resistance features of Acinetobacter baumannii isolates from the ward environment and patients in the burn ICU of a Chinese hospital.

Acinetobacter baumannii is an important opportunistic pathogen that causes severe nosocomial infections, especially in intensive care units (ICUs). Over the past decades, an everincreasing number of hospital outbreaks caused by A. baumannii have been reported worldwide. However, little attention has been directed toward the relationship between A. baumannii isolates from the ward environment and patients in the burn ICU. In this study, 88 A. baumannii isolates (26 from the ward environment and 62 from patients) were collected from the burn ICU of the Southwest Hospital in Chongqing, China, from July through December 2013. Antimicrobial susceptibility testing results showed that drug resistance was more severe in isolates from patients than from the ward environment, with all of the patient isolates being fully resistant to 10 out of 19 antimicrobials tested. Isolations from both the ward environment and patients possessed the β-lactamase genes blaOXA-51, blaOXA-23, blaAmpC, blaVIM, and blaPER. Using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST), these isolates could be clustered into 4 major PFGE types and 4 main sequence types (ST368, ST369, ST195, and ST191) among which, ST368 was the dominant genotype. Epidemiologic and molecular typing data also revealed that a small-scale outbreak of A. baumannii infection was underway in the burn ICU of our hospital during the sampling period. These results suggest that dissemination of β-lactamase genes in the burn ICU might be closely associated with the high-level resistance of A. baumannii, and the ICU environment places these patients at a high risk for nosocomial infection. Cross-contamination should be an important concern in clinical activities to reduce hospitalacquired infections caused by A. baumannii.

Transcriptomic and Metabolomic Analysis Revealed Multifaceted Effects of Phage Protein Gp70.1 on Pseudomonas aeruginosa

The impact of phage infection on the host cell is severe. In order to take over the cellular machinery, some phage proteins were produced to shut off the host biosynthesis early in the phage infection. The discovery and identification of these phage-derived inhibitors have a significant prospect of application in antibacterial treatment. This work presented a phage protein, gp70.1, with non-specific inhibitory effects on Pseudomonas aeruginosa and Escherichia coli. Gp70.1 was encoded by early gene – orf 70.1 from P. aeruginosa phage PaP3. The P. aeruginosa with a plasmid encoding gp70.1 showed with delayed growth and had the appearance of a small colony. The combination of multifaceted analysis including microarray-based transcriptomic analysis, RT-qPCR, nuclear magnetic resonance (NMR) spectroscopy-based metabolomics and phenotype experiments were performed to investigate the effects of gp70.1 on P. aeruginosa. A total of 178 genes of P. aeruginosa mainly involved in extracellular function and metabolism were differentially expressed in the presence of gp70.1 at three examined time points. Furthermore, our results indicated that gp70.1 had an extensive impact on the extracellular phenotype of P. aeruginosa, such as motility, pyocyanin, extracellular protease, polysaccharide, and cellulase. For the metabolism of P. aeruginosa, the main effect of gp70.1 was the reduction of amino acid consumption. Finally, the RNA polymerase sigma factor RpoS was identified as a potential cellular target of gp70.1. Gp70.1 was the first bacterial inhibitor identified from Pseudomonas aeruginosa phage PaP3. It was also the first phage protein that interacted with the global regulator RpoS of bacteria. Our results indicated the potential value of gp70.1 in antibacterial applications. This study preliminarily revealed the biological function of gp70.1 and provided a reference for the study of other phage genes sharing similarities with orf70.1.