Lateral flow assay-based bacterial detection using engineered cell wall binding domains of a phage endolysin

Kong, Minsuk,Shin, Joong Ho,Heu, Sunggi,Park, Je-Kyun,Ryu, Sangryeol Elsevier 2017 Biosensors & bioelectronics Vol.96 No.-

<P><B>Abstract</B></P> <P>The development of a cost-effective and efficient bacterial detection assay is essential for diagnostic fields, particularly in resource-poor settings. Although antibodies have been widely used for bacterial capture, the production of soluble antibodies is still expensive and time-consuming. Here, we developed a nitrocellulose-based lateral flow assay using cell wall binding domains (CBDs) from phage as a recognition element and colloidal gold nanoparticles as a colorimetric signal for the detection of a model pathogenic bacterium, <I>Bacillus cereus (B. cereus)</I>. To improve conjugation efficiency and detection sensitivity, cysteine-glutathione-S-transferase-tagged CBDs and maltose-binding protein-tagged CBDs were produced in <I>Escherichia coli</I> (<I>E. coli</I>) and incorporated in our assays. The sensitivity of the strip to detect <I>B. cereus</I> was 1×10<SUP>4</SUP> CFU/mL and the overall assay time was 20min. The assay showed superior results compared to the antibody-based approach, and did not show any significant cross-reactivity. This proof of concept study indicates that the lateral flow assay using engineered CBDs hold considerable promise as simple, rapid, and cost-effective biosensors for whole cell detection.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The cell wall binding domain (CBD) of endolysin was tested for whole cell detection. </LI> <LI> A lateral flow assay using CBD as a recognition element was developed. </LI> <LI> The assay showed promising results for whole cell detection. </LI> </UL> </P>

LysPBC2, a Novel Endolysin Harboring a <i>Bacillus cereus</i> Spore Binding Domain

Kong, Minsuk,Na, Hongjun,Ha, Nam-Chul,Ryu, Sangryeol American Society for Microbiology 2019 Applied and environmental microbiology Vol.85 No.5

<P>Bacteriophages produce highly evolved lytic enzymes, called endolysins, to lyse peptidoglycan and release their progeny from bacterial cells. Due to their potent lytic activity and specificity, the use of endolysins has gained increasing attention as a natural alternative to antibiotics. Since most endolysins from Gram-positive-bacterium-infecting phages have a modular structure, understanding the function of each domain is crucial to make effective endolysin-based therapeutics. Here, we report the functional and biochemical characterization of a <I>Bacillus cereus</I> phage endolysin, LysPBC2, which has an unusual spore binding domain and a cell wall binding domain. A single point mutation in the spore binding domain greatly enhanced the lytic activity of endolysin at the cost of reduced thermostability. This work contributes to the understanding of the role of each domain in LysPBC2 and will provide insight for the rational design of efficient antimicrobials or diagnostic tools for controlling <I>B. cereus</I>.</P><P>To control the spore-forming human pathogen <I>Bacillus cereus</I>, we isolated and characterized a novel endolysin, LysPBC2, from a newly isolated <I>B. cereus</I> phage, PBC2. Compared to the narrow host range of phage PBC2, LysPBC2 showed very broad lytic activity against all <I>Bacillus</I>, <I>Listeria</I>, and <I>Clostridium</I> species tested. In addition to a catalytic domain and a cell wall binding domain, LysPBC2 has a spore binding domain (SBD) partially overlapping its catalytic domain, which specifically binds to <I>B. cereus</I> spores but not to vegetative cells of <I>B. cereus</I>. Both immunogold electron microscopy and a binding assay indicated that the SBD binds the external region of the spore cortex layer. Several amino acid residues required for catalytic or spore binding activity of LysPBC2 were determined by mutagenesis studies. Interestingly, LysPBC2 derivatives with impaired spore binding activity showed an increased lytic activity against vegetative cells of <I>B. cereus</I> compared with that of wild-type LysPBC2. Further biochemical studies revealed that these LysPBC2 derivatives have lower thermal stability, suggesting a stabilizing role of SBD in LysPBC2 structure.</P><P><B>IMPORTANCE</B> Bacteriophages produce highly evolved lytic enzymes, called endolysins, to lyse peptidoglycan and release their progeny from bacterial cells. Due to their potent lytic activity and specificity, the use of endolysins has gained increasing attention as a natural alternative to antibiotics. Since most endolysins from Gram-positive-bacterium-infecting phages have a modular structure, understanding the function of each domain is crucial to make effective endolysin-based therapeutics. Here, we report the functional and biochemical characterization of a <I>Bacillus cereus</I> phage endolysin, LysPBC2, which has an unusual spore binding domain and a cell wall binding domain. A single point mutation in the spore binding domain greatly enhanced the lytic activity of endolysin at the cost of reduced thermostability. This work contributes to the understanding of the role of each domain in LysPBC2 and will provide insight for the rational design of efficient antimicrobials or diagnostic tools for controlling <I>B. cereus</I>.</P>

Bacteriophage PBC1 and Its Endolysin as an Antimicrobial Agent against <i>Bacillus cereus</i>

Kong, Minsuk,Ryu, Sangryeol American Society for Microbiology 2015 Applied and environmental microbiology Vol.81 No.7

<P><I>Bacillus cereus</I> is an opportunistic human pathogen responsible for food poisoning and other, nongastrointestinal infections. Due to the emergence of multidrug-resistant <I>B. cereus</I> strains, the demand for alternative therapeutic options is increasing. To address these problems, we isolated and characterized a <I>Siphoviridae</I> virulent phage, PBC1, and its lytic enzymes. PBC1 showed a very narrow host range, infecting only 1 of 22 <I>B. cereus</I> strains. Phylogenetic analysis based on the major capsid protein revealed that PBC1 is more closely related to the <I>Bacillus clarkii</I> phage BCJA1c and phages of lactic acid bacteria than to the phages infecting <I>B. cereus</I>. Whole-genome comparison showed that the late-gene region, including the terminase gene, structural genes, and holin gene of PBC1, is similar to that from <I>B. cereus</I> temperate phage 250, whereas their endolysins are different. Compared to the extreme host specificity of PBC1, its endolysin, LysPBC1, showed a much broader lytic spectrum, albeit limited to the genus <I>Bacillus</I>. The catalytic domain of LysPBC1 when expressed alone also showed <I>Bacillus</I>-specific lytic activity, which was lower against the <I>B. cereus</I> group but higher against the <I>Bacillus subtilis</I> group than the full-length protein. Taken together, these results suggest that the virulent phage PBC1 is a useful component of a phage cocktail to control <I>B. cereus</I>, even with its exceptionally narrow host range, as it can kill a strain of <I>B. cereus</I> that is not killed by other phages, and that LysPBC1 is an alternative biocontrol agent against <I>B. cereus</I>.</P>

Experimental identification of pressure drop characteristics at the shell side of an intermediate heat exchanger inside a prototype generation-IV sodium-cooled fast reactor

Kong, Minsuk,Kim, Woo Shik,Chung, Heung June,Choi, HaeSeob,Kim, Joongwoon,Lee, Tae Ho,Euh, Dong-Jin Elsevier 2019 Nuclear engineering and design Vol.348 No.-

<P><B>Abstract</B></P> <P>The flow characteristics at the shell side of a prototype intermediate heat exchanger (p-IHX) in a prototype generation-IV sodium-cooled fast reactor (PGSFR) were investigated experimentally in this study. The shell side of the p-IHX consisted of an inlet window, tube bundles, grid plates, and an exit flow channel. Two different test facilities were newly constructed to identify the pressure drop characteristics due to the complicated configuration with minimized scaling distortion. An intermediate heat exchanger test loop for PGSFR (iHELP) was constructed to characterize the flow resistance of the tube bundle regions with the grid plates and inlet window. The iHELP test section with a slab-shaped cross section was manufactured with a 1/29.6 vol scale ratio by preserving the height of the p-IHX. The hydraulic diameter at the tube bundle regions, the porosity of the grid plates, and the configuration of the tube bundles were conserved. In addition, the Reynolds number was preserved, which determined the flow rate conditions for the iHELP experiments. To investigate the pressure drop characteristics of the exit flow channel (EC), an intermediate heat exchanger test loop for the exit flow channel (IEC) was built with a 1/5 linearly reduced length scale by preserving the geometry of the flow path based on the EC of the p-IHX. The Euler number was conserved with a 1/8 Reynolds number ratio corresponding to a 1/1 flow velocity ratio to the p-IHX for dynamic similarity. By using each test facility the experimental pressure drop data satisfying the experimental uncertainty requirements were obtained separately for the bundle and exit channel regions. The pressure drop at the shell side of the p-IHX under a wide range of flow rate conditions was determined by using the pressure drop results from the iHELP and the IEC test facilities, which would be useful in improving and validating the pressure drop correlations in the IHX design computational code (SHXSA).</P> <P><B>Highlights</B></P> <P> <UL> <LI> Test facility with 1/29.6 volume scale was built for simulating the tube bundle regions. </LI> <LI> Test facility with 1/5 length scale was constructed for simulating the exit flow channel. </LI> <LI> Pressure drop for the IHX shell side was determined by two different test facilities. </LI> <LI> Pressure drop for the IHX was precisely measured over a wide range of flow rates. </LI> </UL> </P>

자연냉각기술이 적용된 BAPV시스템의 실험적 성능평가

공민석(Minsuk Kong),주홍진(Hongjin Joo),곽희열(Heeyoul Kwak) 한국태양에너지학회 2020 한국태양에너지학회 학술대회논문집 Vol.2020 No.6

Fire Image Classification Based on Convolutional Neural Network for Smart Fire Detection

Joohyung Roh(Joohyung Roh),Yukyung Kim(Yukyung Kim),Minsuk Kong(Minsuk Kong) 한국화재소방학회 2022 International Journal of Fire Science and Engineer Vol.36 No.3

This study investigated the effect of the class number on the prediction performance of the convolutional neural network (CNN) classification model that is applied in fire detectors to reduce nuisance fire alarms by appropriately recognizing fire images including those of flames and smoke. A CNN model trained by transfer learning using five image datasets of flame, smoke, normal, haze, and light was realized and trained by altering the class number to generate the classification model. A total of three classification models were generated as follows: classification model 1 was trained using normal and fire images including flames and smoke; classification model 2 was trained using flame, smoke, and normal images; and classification model 3 was trained using flames, smoke, normal, and haze, and light images. A test image dataset independent of training was used to assess the prediction performance of the three classification models. The results indicate that the prediction accuracy for classification models 1, 2, and 3 were approximately 93.0%, 94.2%, and 97.3%, respectively. The performance of the predicted classification improved as the class number increased, because the model could learn with greater precision the features of the normal images that are similar to those of the fire images.

Characterization of LysPBC4, a novel<i>Bacillus cereus</i>-specific endolysin of bacteriophage PBC4

Na, Hongjun,Kong, Minsuk,Ryu, Sangryeol,Millard, Andrew Oxford University Press 2016 FEMS microbiology letters Vol.363 No.12

Specific Detection of Bacillus cereus by Surface Plasmon Resonance Using Phage Endolysin Cell Wall Binding Domains

Jieun SIM,Minsuk KONG,Taejoon KANG,Sangryeol Ryu RYU,Bong Hyun CHUNG 한국생물공학회 2014 한국생물공학회 학술대회 Vol.2014 No.4

이중관 코일형 열교환기에서 나노유체의 열전달 특성에 관한 2상유동 수치해석적 연구

이수민(Sumin Lee),공민석(Minsuk Kong),이승로(Seungro Lee) 대한기계학회 2019 대한기계학회 춘추학술대회 Vol.2019 No.5

Structural Basis for Cell-Wall Recognition by Bacteriophage PBC5 Endolysin

Lee, Ko On,Kong, Minsuk,Kim, Iktae,Bai, Jaewoo,Cha, Soyoung,Kim, Boram,Ryu, Kyoung-Seok,Ryu, Sangryeol,Suh, Jeong-Yong Elsevier 2019 Structure Vol.27 No.9

<P><B>Summary</B></P> <P>Phage endolysins are hydrolytic enzymes that cleave the bacterial cell wall during the lytic cycle. We isolated the bacteriophage PBC5 against <I>Bacillus cereus</I>, a major foodborne pathogen, and describe the molecular interaction between endolysin LysPBC5 and the host peptidoglycan structure. LysPBC5 has an N-terminal glycoside hydrolase 25 domain, and a C-terminal cell-wall binding domain (CBD) that is critical for specific cell-wall recognition and lysis. The crystal and solution structures of CBDs reveal tandem SH3b domains that are tightly engaged with each other. The CBD binds to the peptidoglycan in a bidentate manner via distal β sheet motifs with pseudo 2-fold symmetry, which can explain its high affinity and host specificity. The CBD primarily interacts with the glycan strand of the peptidoglycan layer instead of the peptide crosslink, implicating the tertiary structure of peptidoglycan as the recognition motif of endolysins.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Phage PBC5 encodes a novel endolysin LysPBC5 against <I>Bacillus cereus</I> </LI> <LI> LysPBC5 recognizes host cell walls via a conjoined tandem SH3b repeat fold </LI> <LI> The SH3b repeat fold interacts with peptidoglycans in a bidentate manner </LI> <LI> LysPBC5 primarily binds to glycan strands of the cell-wall structure </LI> </UL> </P> <P><B>Graphical Abstract</B></P> <P>[DISPLAY OMISSION]</P>