Genetic and Biochemical Mechanisms for Bacterial Lipid A Modifiers Associated with Polymyxin Resistance.

Polymyxins are a group of detergent-like antimicrobial peptides that are the ultimate line of defense against carbapenem-resistant pathogens in clinical settings. Polymyxin resistance primarily originates from structural remodeling of lipid A anchored on bacterial surfaces. We integrate genetic, structural, and biochemical aspects of three major types of lipid A modifiers that have been shown to confer intrinsic colistin resistance. Namely, we highlight ArnT, a glycosyltransferase, EptA, a phosphoethanolamine transferase, and the AlmEFG tripartite system, which is restricted to EI Tor biotype of Vibrio cholerae O1. We also discuss the growing family of mobile colistin resistance (MCR) enzymes, each of which is analogous to EptA, and which pose great challenges to global public health.

Defining ICR-Mo, an intrinsic colistin resistance determinant from Moraxella osloensis.

Polymyxin is the last line of defense against severe infections caused by carbapenem-resistant gram-negative pathogens. The emergence of transferable MCR-1/2 polymyxin resistance greatly challenges the renewed interest in colistin (polymyxin E) for clinical treatments. Recent studies have suggested that Moraxella species are a putative reservoir for MCR-1/2 genetic determinants. Here, we report the functional definition of ICR-Mo from M. osloensis, a chromosomally encoded determinant of colistin resistance, in close relation to current MCR-1/2 family. ICR-Mo transmembrane protein was prepared and purified to homogeneity. Taken along with an in vitro enzymatic detection, MALDI-TOF mass spectrometry of bacterial lipid A pools determined that the ICR-Mo enzyme might exploit a possible "ping-pong" mechanism to accept the phosphoethanolamine (PEA) moiety from its donor phosphatidylethanolamine (PE) and then transfer it to the 1(or 4')-phosphate position of lipid A via an ICR-Mo-bound PEA adduct. Structural decoration of LPS-lipid A by ICR-Mo renders the recipient strain of E. coli resistant to polymyxin. Domain swapping assays indicate that the two domains of ICR-Mo cannot be functionally-exchanged with its counterparts in MCR-1/2 and EptA, validating its phylogenetic position in a distinct set of MCR-like genes. Structure-guided functional mapping of ICR-Mo reveals a PE lipid substrate recognizing cavity having a role in enzymatic catalysis and the resultant conference of antibiotic resistance. Expression of icr-Mo in E. coli significantly prevents the formation of reactive oxygen species (ROS) induced by colistin. Taken together, our results define a member of a group of intrinsic colistin resistance genes phylogenetically close to the MCR-1/2 family, highlighting the evolution of transferable colistin resistance.

Mechanistic insights into transferable polymyxin resistance among gut bacteria.

Polymyxins such as colistin are antibiotics used as a final line of defense in the management of infections with multidrug-resistant Gram-negative bacteria. Although natural resistance to polymyxins is rare, the discovery of a mobilized colistin resistance gene (mcr-1) in gut bacteria has raised significant concern. As an intramembrane enzyme, MCR-1 catalyzes the transfer of phosphoethanolamine (PEA) to the 1 (or 4')-phosphate group of the lipid A moiety of lipopolysaccharide, thereby conferring colistin resistance. However, the structural and biochemical mechanisms used by this integral membrane enzyme remain poorly understood. Here, we report the modeled structure of the full-length MCR-1 membrane protein. Together with molecular docking, our structural and functional dissection of the complex of MCR-1 with its phosphatidylethanolamine (PE) substrate suggested the presence of a 12 residue-containing cavity for substrate entry, which is critical for both enzymatic activity and its resultant phenotypic resistance to colistin. More importantly, two periplasm-facing helices (PH2 and PH2') of the trans-membrane domain were essential for MCR-1 activity. MALDI-TOF MS and thin-layer chromatography assays provide both in vivo and in vitro evidence that MCR-1 catalyzes the transfer of PEA from the PE donor substrate to its recipient substrate lipid A. Also, the chemical modification of lipid A species was detected in clinical species of bacteria carrying mcr-1 Our results provide mechanistic insights into transferable MCR-1 polymyxin resistance, raising the prospect of rational design of small molecules that reverse bacterial polymyxin resistance, as a last-resort clinical option to combat pathogens with carbapenem resistance.

Escherichia coli vectors having stringently repressible replication origins allow a streamlining of Crispr/Cas9 gene editing.

Readily curable plasmids facilitate the construction of plasmid-free bacterial strains after the plasmid encoded genes are no longer needed. The most popular of these plasmids features a temperature-sensitive (Ts) pSC101 origin of replication which can readily revert during usage and cannot be used to construct Ts mutations in essential genes. Plasmid pAM34 which contains an IPTG-dependent origin of replication largely overcomes this issue but is limited by carrying the most commonly utilized antibiotic selection and replication origin. This study describes the construction of an expanded series of plasmid vectors having replication origins of p15a, RSF1030 or RSF1031 that like pAM34 have IPTG-dependent replication. Surprisingly, these plasmids can be cured in fewer generations than pAM34. Derivatives of pAM34 with alternative antibiotic selection markers were also constructed. The utility of these vectors is demonstrated in the construction of a CRISPR-Cas9 system consisting of an IPTG-dependent Cas9 plasmid and a curable guide RNA plasmid having a streptomycin counterselection marker. This system was successfully demonstrated by construction of point mutations, deletions and insertions in the E. coli genome with a very high efficiency and in a shorter timescale than extant methods. The plasmids themselves were readily cured either together or singly from the resultant strains with minimal effort.

Coping with inadvertent lysis of Escherichia coli cultures: Strains resistant to lysogeny and infection by the stealthy lysogenic phage Φ80.

Phage Φ80 can infect Escherichia coli in a stealthy manner and persist by forming lysogens. Such Φ80 lysogens are fairly common and often go undetected unless the host is grown at temperatures below 37°C. Since low growth temperatures are required for growing temperature-sensitive mutants and often preferred for large-scale applications such as protein production, Φ80-resistant strains would be useful. We report the construction of E. coli strains that cannot be efficiently lysogenized or infected by bacteriophage Φ80. These strains contain combinations of deletions or mutations in the bacterial attachment site for Φ80 integration and/or deletions in the genes required for phage absorption to the host outer membrane. These strains should help contain and prevent Φ80 infection of E. coli cultures in a laboratory or industrial setting.

Dissemination and Mechanism for the MCR-1 Colistin Resistance.

Polymyxins are the last line of defense against lethal infections caused by multidrug resistant Gram-negative pathogens. Very recently, the use of polymyxins has been greatly challenged by the emergence of the plasmid-borne mobile colistin resistance gene (mcr-1). However, the mechanistic aspects of the MCR-1 colistin resistance are still poorly understood. Here we report the comparative genomics of two new mcr-1-harbouring plasmids isolated from the human gut microbiota, highlighting the diversity in plasmid transfer of the mcr-1 gene. Further genetic dissection delineated that both the trans-membrane region and a substrate-binding motif are required for the MCR-1-mediated colistin resistance. The soluble form of the membrane protein MCR-1 was successfully prepared and verified. Phylogenetic analyses revealed that MCR-1 is highly homologous to its counterpart PEA lipid A transferase in Paenibacili, a known producer of polymyxins. The fact that the plasmid-borne MCR-1 is placed in a subclade neighboring the chromosome-encoded colistin-resistant Neisseria LptA (EptA) potentially implies parallel evolutionary paths for the two genes. In conclusion, our finding provids a first glimpse of mechanism for the MCR-1-mediated colistin resistance.

Pathogenic Streptococcus strains employ novel escape strategy to inhibit bacteriostatic effect mediated by mammalian peptidoglycan recognition protein.

Pathogenic streptococcal species are responsible for some of the most lethal and prevalent animal and human infections. Previous reports have identified a candidate pathogenicity island (PAI) in two highly virulent clinical isolates of Streptococcus suis type 2, a causative agent of high-mortality streptococcal toxic shock syndrome. This PAI contains a type-IVC secretion system C subgroup (type-IVC secretion system) that is involved in the secretion of unknown pathogenic effectors that are responsible for streptococcal toxic shock syndrome caused by highly virulent strains of S. suis. Both virulence protein B4 and virulence protein D4 were demonstrated to be key components of this type-IVC secretion system. In this study, we identify a new PAI family across 3 streptococcal species; Streptococcus genomic island contains type-IV secretion system, which contains a genomic island type-IVC secretion system and a novel PPIase molecule, SP1. SP1 is shown to interact with a component of innate immunity, peptidoglycan recognition protein (PGLYRP-1) and to perturb the PGLYRP-1-mediated bacteriostatic effect by interacting with protein PGLYRP-1. Our study elucidates a novel mechanism by which bacteria escape by components of the innate immune system by secretion of the SP1 protein in pathogenic Streptococci, which then interacts with PGLYRP-1 from the host. Our results provide potential targets for the development of new antimicrobial drugs against bacteria with resistance to innate host immunity.

An Eight-Residue Deletion in Escherichia coli FabG Causes Temperature-Sensitive Growth and Lipid Synthesis Plus Resistance to the Calmodulin Inhibitor Trifluoperazine.

FabG performs the NADPH-dependent reduction of ß-keto acyl-acyl carrier protein substrates in the elongation cycle of fatty acid synthesis. We report the characterization of a temperature-sensitive mutation (fabGΔ8) in Escherichia colifabG that results from an in-frame 8-amino-acid residue deletion in the α6/α7 subdomain. This region forms part of one of the two dimerization interfaces of this tetrameric enzyme and is reported to undergo significant conformational changes upon cofactor binding, which define the entrance to the active-site cleft. The activity of the mutant enzyme is extremely thermolabile and is deficient in forming homodimers at nonpermissive temperatures with a corresponding decrease in fatty acid synthesis both in vivo and in vitro Surprisingly, the fabGΔ8 strain reverts to temperature resistance at a rate reminiscent of that of a point mutant with intragenic pseudorevertants located either on the 2-fold axes of symmetry or at the mouth of the active-site cleft. The fabGΔ8 mutation also confers resistance to the calmodulin inhibitor trifluoperazine and renders the enzyme extremely sensitive to Ca2+in vitro We also observed a significant alteration in the lipid A fatty acid composition of fabGΔ8 strains but only in an lpxC background, probably due to alterations in the permeability of the outer membrane. These observations provide insights into the structural dynamics of FabG and hint at yet another point of regulation between fatty acid and lipid A biosynthesis.IMPORTANCE Membrane lipid homeostasis and its plasticity in a variety of environments are essential for bacterial survival. Since lipid biosynthesis in bacteria and plants is fundamentally distinct from that in animals, it is an ideal target for the development of antibacterial therapeutics. FabG, the subject of this study, catalyzes the first cofactor-dependent reduction in this pathway and is active only as a tetramer. This study examines the interactions responsible for tetramerization through the biochemical characterization of a novel temperature-sensitive mutation caused by a short deletion in an important helix-turn-helix motif. The mutant strain has altered phospholipid and lipid A compositions and is resistant to trifluoperazine, an inhibitor of mammalian calmodulin. Understanding its structural dynamics and its influence on lipid A synthesis also allows us to explore lipid homeostasis as a mechanism for antibiotic resistance.

Brucella BioR regulator defines a complex regulatory mechanism for bacterial biotin metabolism.

The enzyme cofactor biotin (vitamin H or B7) is an energetically expensive molecule whose de novo biosynthesis requires 20 ATP equivalents. It seems quite likely that diverse mechanisms have evolved to tightly regulate its biosynthesis. Unlike the model regulator BirA, a bifunctional biotin protein ligase with the capability of repressing the biotin biosynthetic pathway, BioR has been recently reported by us as an alternative machinery and a new type of GntR family transcriptional factor that can repress the expression of the bioBFDAZ operon in the plant pathogen Agrobacterium tumefaciens. However, quite unusually, a closely related human pathogen, Brucella melitensis, has four putative BioR-binding sites (both bioR and bioY possess one site in the promoter region, whereas the bioBFDAZ [bio] operon contains two tandem BioR boxes). This raised the question of whether BioR mediates the complex regulatory network of biotin metabolism. Here, we report that this is the case. The B. melitensis BioR ortholog was overexpressed and purified to homogeneity, and its solution structure was found to be dimeric. Functional complementation in a bioR isogenic mutant of A. tumefaciens elucidated that Brucella BioR is a functional repressor. Electrophoretic mobility shift assays demonstrated that the four predicted BioR sites of Brucella plus the BioR site of A. tumefaciens can all interact with the Brucella BioR protein. In a reporter strain that we developed on the basis of a double mutant of A. tumefaciens (the ΔbioR ΔbioBFDA mutant), the ß-galactosidase (ß-Gal) activity of three plasmid-borne transcriptional fusions (bioBbme-lacZ, bioYbme-lacZ, and bioRbme-lacZ) was dramatically decreased upon overexpression of Brucella bioR. Real-time quantitative PCR analyses showed that the expression of bioBFDA and bioY is significantly elevated upon removal of bioR from B. melitensis. Together, we conclude that Brucella BioR is not only a negative autoregulator but also a repressor of expression of bioY and bio operons that separately function in biotin transport and the biosynthesis pathway.

Rapid and sensitive detection of H7N9 avian influenza virus by use of reverse transcription-loop-mediated isothermal amplification.

An epidemic of human H7N9 influenza virus infection recently emerged in China whose clinical features include high mortality and which has also resulted in serious economic loss. The novel reassortant avian-origin influenza A (H7N9) virus which was the causative agent of this epidemic raised the possibility of triggering a large-scale influenza pandemic worldwide. It seemed likely that fast molecular detection assays specific for this virus would be in great demand. Here, we report a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method for rapid detection of the hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus, the minimum detection limit of which was evaluated using in vitro RNA transcription templates. In total, 135 samples from clinical specimens (from either patients or poultry) were tested using this method in comparison with the real-time PCR recommended by the World Health Organization (WHO). Our results showed that (i) RT-LAMP-based trials can be completed in approximately 12 to 23 min and (ii) the detection limit for the H7 gene is around 10 copies per reaction, similar to that of the real-time PCR, whereas the detection limit for its counterpart the N9 gene is 5 copies per reaction, a 100-fold-higher sensitivity than the WHO-recommended method. Indeed, this excellent performance of our method was also validated by the results for a series of clinical specimens. Therefore, we believe that the simple, fast, and sensitive method of RT-LAMP might be widely applied for detection of H7N9 infections and may play a role in prevention of an influenza pandemic.

Action and mechanism of the colistin resistance enzyme MCR-4.

Colistin is the last-resort antibiotic against lethal infections with multidrug-resistant bacterial pathogens. A rainbow coalition of mobile colistin resistance (mcr) genes raises global health concerns. Here, we describe the action and mechanism of colistin resistance imparted by MCR-4, a recently-identified member from the broader MCR family. We found that MCR-4 originates from the silenced variant of Shewanella frigidimarina via progressive evolution and forms a phylogenetically-distinct group from the well-studied MCR-1/2 family. Domain-swapping experiments further confirmed that MCR-1 and MCR-4 transmembrane and catalytic domains are not functionally-interchangeable. However, structural and functional analyses demonstrated that MCR-4 possesses a similar PE lipid substrate-recognizable cavity and exploits an almost-identical ping-pong catalysis mechanism. MCR-4 also can alleviate colistin-triggered accumulation of reactive oxygen species (ROS). Taken together, this finding constitutes a functional proof that MCR-4 proceeds in a distinct evolutionary path to fulfill a consistent molecular mechanism, resulting in phenotypic colistin resistance.

A Genomic, Evolutionary, and Mechanistic Study of MCR-5 Action Suggests Functional Unification across the MCR Family of Colistin Resistance.

A growing number of mobile colistin resistance (MCR) proteins is threatening the renewed interest of colistin as a "last-resort" defense against carbapenem-resistant pathogens. Here, the comparative genomics of a large plasmid harboring mcr-5 from Aeromonas hydrophila and the structural/functional perspectives of MCR-5 action are reported. Whole genome sequencing has identified the loss of certain parts of the Tn3-type transposon typically associated with mcr-5, providing a clue toward its mobilization. Phylogeny of MCR-5 suggests that it is distinct from the MCR-1/2 sub-lineage, but might share a common ancestor of MCR-3/4. Domain-swapping analysis of MCR-5 elucidates that its two structural motifs (transmembrane domain and catalytic domain) are incompatible with its counterparts in MCR-1/2. Like the rest of the MCR family, MCR-5 exhibits a series of conservative features, including zinc-dependent active sites, phosphatidylethanolamine-binding cavity, and the mechanism of enzymatic action. In vitro and in vivo evidence that MCR-5 catalyzes the addition of phosphoethanolamine to the suggestive 4'-phosphate of lipid A moieties is integrated, and results in the consequent polymyxin resistance. In addition, MCR-5 alleviates the colistin-induced formation of reactive oxygen species in E. coli. Taken together, the finding suggests that a growing body of MCR family resistance enzymes are functionally unified.

An Evolutionarily Conserved Mechanism for Intrinsic and Transferable Polymyxin Resistance.

Polymyxins, a family of cationic antimicrobial cyclic peptides, act as a last line of defense against severe infections by Gram-negative pathogens with carbapenem resistance. In addition to the intrinsic resistance to polymyxin E (colistin) conferred by Neisseria eptA, the plasmid-borne mobilized colistin resistance gene mcr-1 has been disseminated globally since the first discovery in Southern China, in late 2015. However, the molecular mechanisms for both intrinsic and transferable resistance to colistin remain largely unknown. Here, we aim to address this gap in the knowledge of these proteins. Structural and functional analyses of EptA and MCR-1 and -2 have defined a conserved 12-residue cavity that is required for the entry of the lipid substrate, phosphatidylethanolamine (PE). The in vitro and in vivo data together have allowed us to visualize the similarities in catalytic activity shared by EptA and MCR-1 and -2. The expression of either EptA or MCR-1 or -2 is shown to remodel the surface of enteric bacteria (e.g., Escherichia coli, Salmonella enterica, Klebsiella pneumoniae, etc.), rendering them resistant to colistin. The parallels in the PE substrate-binding cavities among EptA, MCR-1, and MCR-2 provide a comprehensive understanding of both intrinsic and transferable colistin resistance. Domain swapping between EptA and MCR-1 and -2 reveals that the two domains (transmembrane [TM] region and phosphoethanolamine [PEA] transferase) are not functionally exchangeable. Taken together, the results represent a common mechanism for intrinsic and transferable PEA resistance to polymyxin, a last-resort antibiotic against multidrug-resistant pathogens.IMPORTANCE EptA and MCR-1 and -2 remodel the outer membrane, rendering bacteria resistant to colistin, a final resort against carbapenem-resistant pathogens. Structural and functional analyses of EptA and MCR-1 and -2 reveal parallel PE lipid substrate-recognizing cavities, which explains intrinsic and transferable colistin resistance in gut bacteria. A similar mechanism is proposed for the catalytic activities of EptA and MCR-1 and -2. Together, they constitute a common mechanism for intrinsic and transferable polymyxin resistance.

Spread of MCR-3 Colistin Resistance in China: An Epidemiological, Genomic and Mechanistic Study.

BACKGROUND: Mobilized resistance to colistin is evolving rapidly and its global dissemination poses a severe threat to human health and safety. Transferable colistin resistance gene, mcr-3, first identified in Shandong, China, has already been found in several countries in multidrug-resistant human infections. Here we track the spread of mcr-3 within 13 provinces in China and provide a complete characterization of its evolution, structure and function. METHODS: A total of 6497 non-duplicate samples were collected from thirteen provinces in China, from 2016 to 2017 and then screened for the presence of mcr-3 gene by PCR amplification. mcr-3-positive isolates were analyzed for antibiotic resistance and by southern blot hybridization, transfer analysis and plasmid typing. We then examined the molecular evolution of MCR-3 through phylogenetic analysis. Furthermore, we also characterized the structure and function of MCR-3 through circular dichroism analyses, inductively coupled plasma mass spectrometry (ICP-MS), liquid chromatography mass spectrometry (LC/MS), confocal microscopy and chemical rescue tests. FINDINGS: 49 samples (49/6497 = 0.75%) were mcr-3 positive, comprising 40 samples (40/4144 = 0.97%) from 2017 and 9 samples (9/2353 = 0.38%) from 2016. Overall, mcr-3-positive isolates were distributed in animals and humans in 8 of the 13 provinces. Three mcr-3-positive IncP-type and one mcr-1-bearing IncHI2-like plasmids were identified and characterized. MCR-3 clusters with PEA transferases from Aeromonas and other bacteria and forms a phylogenetic entity that is distinct from the MCR-1/2/P(M) family, the largest group of transferable colistin resistance determinants. Despite that the two domains of MCR-3 not being exchangeable with their counterparts in MCR-1/2, structure-guided functional mapping of MCR-3 defines a conserved PE-lipid recognizing cavity prerequisite for its enzymatic catalysis and its resultant phenotypic resistance to colistin. We therefore propose that MCR-3 uses a possible "ping-pong" mechanism to transfer the moiety of PEA from its donor PE to the 1(or 4')-phosphate of lipid A via an adduct of MCR-3-bound PEA. Additionally, the expression of MCR-3 in E. coli prevents the colistin-triggered formation of reactive oxygen species (ROS) and interferes bacterial growth and viability. INTERPRETATION: Our results provide an evolutionary, structural and functional definition of MCR-3 and its epidemiology in China, paving the way for smarter policies, better surveillance and effective treatments.

Deciphering MCR-2 Colistin Resistance.

Antibiotic resistance is a prevalent problem in public health worldwide. In general, the carbapenem ß-lactam antibiotics are considered a final resort against lethal infections by multidrug-resistant bacteria. Colistin is a cationic polypeptide antibiotic and acts as the last line of defense for treatment of carbapenem-resistant bacteria. Very recently, a new plasmid-borne colistin resistance gene, mcr-2, was revealed soon after the discovery of the paradigm gene mcr-1, which has disseminated globally. However, the molecular mechanisms for MCR-2 colistin resistance are poorly understood. Here we show a unique transposon unit that facilitates the acquisition and transfer of mcr-2 Evolutionary analyses suggested that both MCR-2 and MCR-1 might be traced to their cousin phosphoethanolamine (PEA) lipid A transferase from a known polymyxin producer, Paenibacillus Transcriptional analyses showed that the level of mcr-2 transcripts is relatively higher than that of mcr-1 Genetic deletions revealed that the transmembrane regions (TM1 and TM2) of both MCR-1 and MCR-2 are critical for their location and function in bacterial periplasm, and domain swapping indicated that the TM2 is more efficient than TM1. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) confirmed that all four MCR proteins (MCR-1, MCR-2, and two chimeric versions [TM1-MCR-2 and TM2-MCR-1]) can catalyze chemical modification of lipid A moiety anchored on lipopolysaccharide (LPS) with the addition of phosphoethanolamine to the phosphate group at the 4' position of the sugar. Structure-guided site-directed mutagenesis defined an essential 6-residue-requiring zinc-binding/catalytic motif for MCR-2 colistin resistance. The results further our mechanistic understanding of transferable colistin resistance, providing clues to improve clinical therapeutics targeting severe infections by MCR-2-containing pathogens.IMPORTANCE Carbapenem and colistin are the last line of refuge in fighting multidrug-resistant Gram-negative pathogens. MCR-2 is a newly emerging variant of the mobilized colistin resistance protein MCR-1, posing a potential challenge to public health. Here we report transfer of the mcr-2 gene by a unique transposal event and its possible origin. Distribution of MCR-2 in bacterial periplasm is proposed to be a prerequisite for its role in the context of biochemistry and the colistin resistance. We also define the genetic requirement of a zinc-binding/catalytic motif for MCR-2 colistin resistance. This represents a glimpse of transferable colistin resistance by MCR-2.

Determination of Scrub typhus Suggests a New Epidemic Focus in the Anhui Province of China.

In 2007, 19 cases of a scrub typhus epidemic occurred within a week at a sports school in Mingguang County, Anhui Province, where no previous incidence of this mite borne disease had been reported. Sero-surveillance in 2009 indicated that 10 of the 100 school students possessed anti-Orientia tsutsugamushi antibodies. From 2009 to 2013, 60 small animals and 2250 mites were collected in the vicinity of the school. 5 of the Apodemus agrarius samples and 1 group of Leptotrombidium linhuaikongense tested positive via PCR for O. tsutsugamushi. Two strains of O. tsutsugamushi were identified by injecting Kun Ming (KM) mice peritoneally with the organs of either Apodemus agrarius or Leptotrombidium linhuaikongense. Apart from sharing 98% homology with the O. tsutsugamushi Yongworl strain, genes encoding the membrane protein from the two O. tsutsugamushi isolates shared >99% sequence homology with each other, reflecting the consistency of the pathogen in both the vector and the host. In addition, we also characterized a chronic scrub typhus infection in a local patient. The membrane protein gene fragment from the patient's blood shared 99% homology with O. tsutsugamushi Gilliam strain, suggesting that more than one O. tsutsugamushi strain is present at this location.

Immunisation With Immunodominant Linear B Cell Epitopes Vaccine of Manganese Transport Protein C Confers Protection against Staphylococcus aureus Infection.

Vaccination strategies for Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA) infections have attracted much research attention. Recent efforts have been made to select manganese transport protein C, or manganese binding surface lipoprotein C (MntC), which is a metal ion associated with pathogen nutrition uptake, as potential candidates for an S. aureus vaccine. Although protective humoral immune responses to MntC are well-characterised, much less is known about detailed MntC-specific B cell epitope mapping and particularly epitope vaccines, which are less-time consuming and more convenient. In this study, we generated a recombinant protein rMntC which induced strong antibody response when used for immunisation with CFA/IFA adjuvant. On the basis of the results, linear B cell epitopes within MntC were finely mapped using a series of overlapping synthetic peptides. Further studies indicate that MntC113-136, MntC209-232, and MntC263-286 might be the original linear B-cell immune dominant epitope of MntC, furthermore, three-dimensional (3-d) crystal structure results indicate that the three immunodominant epitopes were displayed on the surface of the MntC antigen. On the basis of immunodominant MntC113-136, MntC209-232, and MntC263-286 peptides, the epitope vaccine for S. aureus induces a high antibody level which is biased to TH2 and provides effective immune protection and strong opsonophagocytic killing activity in vitro against MRSA infection. In summary, the study provides strong proof of the optimisation of MRSA B cell epitope vaccine designs and their use, which was based on the MntC antigen in the development of an MRSA vaccine.

Deoxynivalenol inhibits proliferation and induces apoptosis in human umbilical vein endothelial cells.

Deoxynivalenol (DON) is a stable mycotoxins found in cereals infected by certain fungal species and causes adverse health effects in animals and human such as vomiting, diarrhea and reproductive toxicity. In this study, we investigated the toxic and apoptotic effects of DON in human umbilical vein endothelial cells (HUVECs), a good model for studying inflammation. The results show that DON significantly inhibited the viability of HUVECs. DON could also inhibit the proliferation of HUVECs through G2/M phase arrest in cell cycle progression. Moreover, oxidative stress induced by DON was indicated by observations of increased levels of reactive oxygen species (ROS). In addition, DON also causes mitochondrial damage by decreasing the mitochondrial membrane potential and inducing apoptosis by up-regulation of apoptosis-related genes like caspase-3, caspase-9, and Bax genes, and down-regulation of Bcl-2 gene. These results together suggest that DON could induce cell cycle arrest, oxidative stress, and apoptosis in HUVECs.

Genome-wide miRNA-profiling of aflatoxin B1-induced hepatic injury using deep sequencing.

Aflatoxin B1 is a potent carcinogen which can induce** hepatocellular carcinoma (HCC) in mammals. Though microRNAs are known to play important roles in tumorigenesis, the functional complexity of microRNAs in AFB1-induced hepatocellular tumorigenesis has not yet been elucidated. Here, we applied Illumina deep sequencing technology for high-throughput profiling of microRNA in rat liver tissue before and after treatment with aflatoxin B1. Analysis of mature miRNAs from different arms of pre-miRNAs allowed us to identify the predominant form of miRNA. We studied the differential expression profile of miRNAs in two libraries, identifying several cancer-related microRNAs which exhibit abnormal expression. KEGG analysis indicated that predicted target genes of differentially expressed miRNAs are involved in cancer-related pathways. Bioinformatic analysis predicted 16 potential novel miRNAs. Our work provides new insights at the miRNA level into AFB1-induced hepatic injury which may lead to HCC.