Hyunjin Yoon

Identification and Characterization of Outer Membrane Vesicle-Associated Proteins in Salmonella enterica Serovar Typhimurium

ABSTRACT Salmonella enterica serovar Typhimurium is a primary cause of enteric diseases and has acquired a variety of virulence factors during its evolution into a pathogen. Secreted virulence factors interact with commensal flora and host cells and enable Salmonella to survive and thrive in hostile environments. Outer membrane vesicles (OMVs) released from many Gram-negative bacteria function as a mechanism for the secretion of complex mixtures, including virulence factors. We performed a proteomic analysis of OMVs that were isolated under standard laboratory and acidic minimal medium conditions and identified 14 OMV-associated proteins that were observed in the OMV fraction isolated only under the acidic minimal medium conditions, which reproduced the nutrient-deficient intracellular milieu. The inferred roles of these 14 proteins were diverse, including transporter, enzyme, and transcriptional regulator. The absence of these proteins influenced Salmonella survival inside murine macrophages. Eleven of these proteins were predicted to possess secretion signal sequences at their N termini, and three (HupA, GlnH, and PhoN) of the proteins were found to be translocated into the cytoplasm of host cells. The comparative proteomic profiling of OMVs performed in this study revealed different protein compositions in the OMVs isolated under the two different conditions, which indicates that the OMV cargo depends on the growth conditions and provides a deeper insight into how Salmonella utilizes OMVs to adapt to environmental changes.

hfq Plays Important Roles in Virulence and Stress Adaptation in Cronobacter sakazakii ATCC 29544

ABSTRACT Cronobacter spp. are opportunistic pathogens that cause neonatal meningitis and sepsis with high mortality in neonates. Despite the peril associated with Cronobacter infection, the mechanisms of pathogenesis are still being unraveled. Hfq, which is known as an RNA chaperone, participates in the interaction with bacterial small RNAs (sRNAs) to regulate posttranscriptionally the expression of various genes. Recent studies have demonstrated that Hfq contributes to the pathogenesis of numerous species of bacteria, and its roles are varied between bacterial species. Here, we tried to elucidate the role of Hfq in C. sakazakii virulence. In the absence of hfq, C. sakazakii was highly attenuated in dissemination in vivo, showed defects in invasion (3-fold) into animal cells and survival (103-fold) within host cells, and exhibited low resistance to hydrogen peroxide (102-fold). Remarkably, the loss of hfq led to hypermotility on soft agar, which is contrary to what has been observed in other pathogenic bacteria. The hyperflagellated bacteria were likely to be attributable to the increased transcription of genes associated with flagellar biosynthesis in a strain lacking hfq. Together, these data strongly suggest that hfq plays important roles in the virulence of C. sakazakii by participating in the regulation of multiple genes.

Endolysin LysSA97 is synergistic with carvacrol in controlling Staphylococcus aureus in foods

LysSA97 is an endolysin encoded by the bacteriophage SA97, the genome sequence of which has been recently revealed. LysSA97 has lytic activity against a variety of Staphylococcus strains that cause foodborne illness. In order to improve its potential as a biocontrol agent against Staphylococcus, various types of essential oil-derived active compounds were tested in combination with LysSA97; carvacrol exhibited significant synergistic effects when combined with LysSA97. The synergistic antimicrobial activity between endolysin and carvacrol in food products, including milk and beef, were investigated. While LysSA97 (376nM) and carvacrol (3.33mM) showed 0.8±0.2 and 1.0±0.0logCFU/mL reduction in Staphylococcus aureus cells, respectively; when applied alone in bacterial culture, the cocktail containing both at the same concentrations exhibited a bacterial decrease of 4.5±0.2logCFU/mL. The synergistic activity of carvacrol was also reproduced in combination with other endolysins, and their cooperative bactericidal effects were validated in ten additional S. aureus strains, including two methicillin-resistant S. aureus (MRSA), suggesting the wide application of carvacrol as a bactericidal agent coupled with endolysin. When LysSA97 and carvacrol were used in combination in foods, the synergistic activity appeared to be influenced by the total lipid content of foods, and bacteria in skim milk were more drastically inactivated than those in whole milk. Therefore, this is the first report demonstrating that endolysin and carvacrol act synergistically to inactivate S. aureus in food products.

Zwitterionic sulfobetaine polymer-immobilized surface by simple tyrosinase-mediated grafting for enhanced antifouling property

Introducing antifouling property to biomaterial surfaces has been considered an effective method for preventing the failure of implanted devices. In order to achieve this, the immobilization of zwitterions on biomaterial surfaces has been proven to be an excellent way of improving anti-adhesive potency. In this study, poly(sulfobetaine-co-tyramine), a tyramine-conjugated sulfobetaine polymer, was synthesized and simply grafted onto the surface of polyurethane via a tyrosinase-mediated reaction. Surface characterization by water contact angle measurements, X-ray photoelectron spectroscopy and atomic force microscopy demonstrated that the zwitterionic polymer was successfully introduced onto the surface of polyurethane and remained stable for 7days. In vitro studies revealed that poly(sulfobetaine-co-tyramine)-coated surfaces dramatically reduced the adhesion of fibrinogen, platelets, fibroblasts, and S. aureus by over 90% in comparison with bare surfaces. These results proved that polyurethane surfaces grafted with poly(sulfobetaine-co-tyramine) via a tyrosinase-catalyzed reaction could be promising candidates for an implantable medical device with excellent bioinert abilities.nnnSTATEMENT OF SIGNIFICANCEnAntifouling surface modification is one of the key strategy to prevent the thrombus formation or infection which occurs on the surface of biomaterial after transplantation. Although there are many methods to modify the surface have been reported, necessity of simple modification technique still exists to apply for practical applications. The purpose of this study is to modify the biomaterials surface by simply immobilizing antifouling zwitterion polymer via enzyme tyrosinase-mediated reaction which could modify versatile substrates in mild aqueous condition within fast time period. After modification, pSBTA grafted surface becomes resistant to various biological factors including proteins, cells, and bacterias. This approach appears to be a promising method to impart antifouling property on biomaterial surfaces.

Transcriptional response of selected genes of Salmonella enterica serovar Typhimurium biofilm cells during inactivation by superheated steam

Superheated steam (SHS), produced by the addition of heat to saturated steam (SS) at the same pressure, has great advantages over conventional heat sterilization due to its high temperature and accelerated drying rate. We previously demonstrated that treatment with SHS at 200°C for 10 sec inactivated Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes biofilm cells on the surface of stainless steel to below the detection limit. However, bacteria withstanding heat stress become more resistant to other stress conditions, and may be more virulent when consumed by a host. Herein, we studied the transcriptional regulation of genes important for stress resistance and virulence in Salmonella biofilms after SHS treatments. Genes encoding heat shock proteins and general stress resistance proteins showed transcriptional surges after 1 sec of SHS treatment at 200°C, with parallel induction of stress-related regulator genes including rpoE, rpoS, and rpoH. Interestingly, Salmonella biofilm cells exposed to SHS showed decreased transcription of flagella and Salmonella pathogenicity island-1 (SPI-1) genes required for motility and invasion of host cells, respectively, whereas increased transcription of SPI-2 genes, important for bacterial survival and replication inside host cells, was detected. When the transcriptional response was compared between cells treated with SHS (200°C) and SS (100°C), SHS caused immediate changes in gene expression by shorter treatments. Understanding the status of Salmonella virulence and stress resistance induced by SHS treatments is important for wider application of SHS in controlling Salmonella biofilm formation during food production.

The complete genome sequence of Cronobacter sakazakii ATCC 29544 T , a food-borne pathogen, isolated from a child’s throat

BackgroundCronobacter sakazakii is an emerging opportunistic pathogen that is associated with rare but life-threatening cases of severe diseases: meningitis, necrotizing enterocolitis, and sepsis in premature and full-term infants. However, the pathogenesis mechanism of this pathogen remains largely unknown. To determine its pathogenesis at the genomic level, the genome of C. sakazakii ATCC 29544T was completely sequenced and analyzed.ResultsThe genomic DNA, containing a circular chromosome and three plasmids, is composed of 4,511,265xa0bp with a GC content of 56.71%, containing 4380 predicted open reading frames (ORFs), 22 rRNA genes, and 83 tRNA genes. The plasmids, designated pCSK29544_p1, pCSK29544_p2, and pCSK29544_p3, were 93,905-bp, 4938-bp, and 53,457-bp with GC contents of 57.02, 54.88, and 50.07%, respectively. They were also predicted to have 72, 6, and 57 ORFs without RNA genes.ConclusionsThe strain ATCC 29544T genome has ompA and ibeB-homologous cusC genes, probably associated with the invasion of human brain microvascular endothelial cells (BMECs). In addition, gene clusters for siderophore production (iucABCD/iutA) and the related transport system (eitCBAD) were detected in pCSK29544_p1 plasmid, indicating better iron uptake ability for survival. Furthermore, to survive under extremely dry condition like milk powder, this genome has gene clusters for biosynthesis of capsular proteins (CSK29544_00281-00284) and cellulose (CSK29544_01124-01127) for biofilm formation and a gene cluster for utilization of sialic acid in the milk (nanKTAR). The genome information of C. sakazakii ATCC 29544T would provide further understanding of its pathogenesis at the molecular level for the regulation of pathogenicity and the development of a rapid detection method using biomarkers.

New antibacterial-core structures based on styryl quinolinium

Quaternary quinolinium salts have been widely used as alternative antimicrobial agents. In an effort to improve the current quinolinium compounds and determine the relation between antibacterial activity and substituted functional groups, 10 different styryl quinolinium derivatives with various quaternary ammonium electron acceptors, electron donors, and counter anions were rationally designed. Among the 10 styryl quinoliniums, six compounds exhibited bactericidal effects against Gram-positive bacteria, with minimum inhibitory concentrations (MICs) of 2.4–37.5 μg/mL. In addition, two compounds, namely DA-DMQ1,4-T and DA-DMQ1,4-TMS, showed low MICs of 18.75–75 μg/mL with Gram-negative bacteria. In general, compounds possessing electron acceptor groups with a strong electron-withdrawing ability exhibited high bactericidal activity against diverse bacterial species. Co-administration of quinolinium (1.17–9.36 μg/mL) and broad-spectrum β-lactam antibiotic ampicillin (0.02–2.34 μg/mL) showed synergistic bactericidal effects on both Gram-positive and Gramnegative bacteria. This study provides guidelines for the development of new quinolinium salts with a prominent antimicrobial activity.

Temporal regulation of Salmonella pathogenicity Island 1 (SPI-1) hilA by Hfq in Salmonella enterica serovar typhimurium

Hfq is a global regulatory RNA-binding protein abundant in many bacteria. In terms of virulence regulation in Salmonella, Hfq binds to and stabilizes the mRNA of hilD, a master regulator of Salmonella pathogenicity island 1 (SPI-1), and Hfq activates SPI-1 gene transcription during the late exponential-growth phase. In contrast to the positive roles of Hfq in SPI-1 regulation, we observed that the absence of Hfq increased the expression of SPI-1 regulators, including HilA and InvF, during the late stationary phase. This result suggests that Hfq has pleiotropic functions, controlling SPI-1 positively or negatively depending on the growth conditions.

New virulence factor CSK29544_02616 as LpxA binding partner in Cronobacter sakazakii

Cronobacter sakazakii is an opportunistic pathogen that can cause meningitis and necrotizing enterocolitis in premature infants, but its virulence determinants remain largely unknown. In this study, a transposon-mediated random-mutant library of C. sakazakii was used to identify new virulence factors. Compared to wild-type bacteria, a mutant lacking CSK29544_02616 (referred to as labp) was defective in invasion into intestinal epithelial cells (by at least 1000-fold) and showed less phagocytosis by macrophages (by at least 50-fold). The lack of labp in C. sakazakii changed the profile of outer membrane proteins, decreased the production of lipopolysaccharides, and increased the production of membrane phospholipids. Bacterial physiological characteristics including surface hydrophobicity and motility were also altered in the absence of labp, presumably because of changes in the bacterial-envelope structure. To systematically determine the role of labp, ligand fishing was conducted using Labp as a bait, which revealed LpxA as a binding partner of Labp. LpxA is UDP-N-acetylglucosamine (GlcNAc) acyltransferase, the first enzyme in the pathway of lipid A biosynthesis. Labp increased the enzymatic activity of LpxA without influencing lpxA expression. Considering multifaceted roles of lipopolysaccharides in virulence regulation, Labp is a novel virulence factor that promotes the production of lipid A by LpxA in Cronobacter.

Salmonella Typhimurium Sensing Strategy Based on the Loop-Mediated Isothermal Amplification Using Retroreflective Janus Particle as a Nonspectroscopic Signaling Probe

Loop-mediated isothermal amplification (LAMP) is a powerful gene amplification method, which has many advantages, including high specificity, sensitivity, and simple operation. However, quantitative analysis of the amplified target gene with the LAMP assay is very difficult. To overcome this limitation, we developed a novel biosensing platform for molecular diagnosis by integrating the LAMP method and retroreflective Janus particle (RJP) together. The final amplified products of the LAMP assay are dumbbell-shaped DNA structures, containing a single-stranded loop with two different sequences. Therefore, the concentration of the amplified products can be measured in a manner similar to the sandwich-type immunoassay. To carry out the sandwich-type molecular diagnostics using the LAMP product, two DNA probes, with complementary sequences to the loop-regions, were prepared and immobilized on both the sensing surface and the surface of the RJPs. When the amplified LAMP product was applied to the sensing surface, the surface-immobilized DNA probe hybridized to the loop-region of the LAMP product to form a double-stranded structure. When the DNA probe-conjugated RJPs were injected, the RJPs bound to the unreacted loop-region of the LAMP product. The number of RJPs bound to the loop-region of the LAMP product was proportional to the concentration of the amplified LAMP product, indicating that the concentration of the target gene can be quantitatively analyzed by counting the number of observed RJPs. Using the developed system, a highly sensitive and selective quantification of Salmonella was successfully performed with a detection limit of 102 CFU.