• No results found

Characteristics of outer-membrane vesiclesderived from klebsiella pneumoniae atcc 13883

N/A
N/A
Protected

Academic year: 2020

Share "Characteristics of outer-membrane vesiclesderived from klebsiella pneumoniae atcc 13883"

Copied!
6
0
0

Loading.... (view fulltext now)

Full text

(1)

*Corresponding author:Sung Hee Hyun

Department of Senior Healthcare, BK21 plus program, Graduated School, Eulji University,

INTERNATIONAL JOURNAL OF CURRENT MEDICAL AND

PHARMACEUTICAL RESEARCH

ISSN: 2395-6429, Impact Factor: 4.656

Available Online at www.journalcmpr.com

Volume 4; Issue 3(A); March 2018; Page No. 3171-3176

DOI: http://dx.doi.org/10.24327/23956429.ijcmpr20180419

Research Article

CHARACTERISTICS OF OUTER-MEMBRANE VESICLES DERIVED FROM KLEBSIELLA

PNEUMONIAE ATCC 13883

Hee Sang You

1,2

., Min Joon Kim

1

., Yeon Jeong Ok

1

., Eun Jeoung Lee

3

.,

Sang Sun Kang

3

and Sung Hee Hyun

1,2

*

1

Department of Biomedical Laboratory Science, Eulji University, 77, Gyeryong-ro,

771 beon-gil, Jung-gu, Daejeon, 34824, Republic of Korea

2

Department of Senior Healthcare, BK21 plus program, Graduated School, Eulji University, 77,

Gyeryong-ro, 771 beon-gil, Jung-gu, Daejeon, 34824, Republic of Korea

3

Department of Biology Education, Chungbuk National University, 1, Chundae-ro, Seowon-gu,

Cheongju, Chungbuk, 28644, Republic of Korea

ARTICLE INFO ABSTRACT

Many Gram-negative bacteria secrete outer-membrane vesicles (OMVs) into their surrounding environment. OMVs deliver effect or molecules to nearby cells and play important roles in bacterial survival and pathogenesis. Klebsiella pneumoniae is a prominent opportunistic pathogen, but its production of OMVs and the roles that OMVs play in its biology have not been clearly elucidated. This study examined the secretion and protein profiles of K. pneumoniae OMVs. K. pneumoniae ATCC 13883 secreted OMVs into the extracellular environment. The OMVs were associated with 159 different proteins, including many outer-membrane proteins and relatively few inner-membrane proteins. They also included proteins involved in the production of vesicles, the elimination of toxins and phages, the intercellular transport of genetic material and proteins, the targeting of host cells, and the modulation of host defenses. This study will help to reveal the functions of OMVs in K. pneumoniae in order to generate new vaccines and therapies for multi-drug resistant K. pneumoniae strains.

Copyright © 2018 'Sung Hee Hyun et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

INTRODUCTION

Many bacteria, including Klebsiella pneumoniae, produce and secrete outer-membrane vesicles (OMVs) under normal conditions. Bacterial OMVs regulate intercellular signaling and DNA and protein transfer and have been reported to cause cell death1. OMVs are important vehicles for the simultaneous delivery of many effect or molecules, including toxins, to host cells2 and competitors3. In addition, OMVs may be used as vectors to deliver vaccines and other therapeutic compounds. OMVs are composed of outer-membrane, periplasmic, and cytoplasmic proteins; outer-membrane lipids; lipopolysaccharides (LPSs); DNA; RNA; cell-cell signaling molecules; and virulence factors4. The OMVs of pathogenic Gram-negative bacteria are associated with avariety of toxinsand virulence factors, including the heat-labile toxin and cytolethal distending toxin of Campylobacter jejuni5; ClyA of Escherichia coli6; β-lactamases, hemolytic phospholipase C, and alkaline phosphatases of Pseudomonas aeruginosa7; and VacA of Helicobacter pylori8. Universal virulence mechanisms

facilitate host colonization and the saprophytic biology that occurs during infection9. The virulence factors and other pathogen-associated molecular patterns (PAMPs) in OMVs are targeted to host cells, in which they can induce pathology and modulate defense mechanisms. In addition to extracellular toxins, lytic proteins, and other host-targeted molecules, virulence mechanisms involve proteins that facilitatethe acquisition of trace elements and also the molecular complexes that participate in secretion.

K. pneumoniae is a respiratory pathogen that causes substantial morbidity and mortality. Clinical isolates of K. pneumoniae typically exhibit multi-drug resistance, causing serious problems for the treatment of infections10. AlthoughK. Pneumoniae produces numerous virulence factors; including capsular polysaccharides11, LPS12, side rophores13, and adhesins; the specific factors that are cytotoxic to host cells have not yet been identified. The capsular polysaccharide of K. pneumoniae has been related to virulence in host cells, but its expression level in different strains is not directly related to the virulence of the strains, and the cytotoxicity of K. pneumoniae

Article History:

Received 9th December, 2017 Received in revised form 21st January, 2018

Accepted 05th February, 2018 Published online 28th March, 2018

Key words:

(2)

International Journal Of Current Medical And Pharmaceutical Research

is not directly associated with bacterial adherence to epit cells14. The secretion of toxic extracellular complexes from pneumoniae has been shown to be responsible for causing damage to lung tissues and to be related to

virulence15. We speculate that the toxic extracellular complexes produced by K. pneumoniae may be

induce cytotoxicity in host cells.

The OMVs produced by bacteria during normal growth are essential for survival and pathogenesis during host infection Recent studies have revealed some of the divers

OMVs; however, the mechanisms by which OMVs are formed and acquire specific proteins, as well as the ultimate functions of the OMVs, remain to be elucidated. In this study, we investigated the production and proteome composition of OMVs produced by K. pneumoniae ATCC 13883

MATERIALS AND METHODS

Cell culture and purification of OMVs

We purchased K. pneumoniae ATCC

American Type Culture Collection. We cultured the cells in Luria-Bertani (LB) medium (Difco, Leeuwarden, Netherlands) at 37°C. We purified OMVs from supernatants of bacterial cultures as described previously 9. We transferred a single colony of K. pneumoniae ATCC 13883 to 5 mL LB broth (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0) and incubated the culture for 8 hat 37°Cwith shaking at 150 rpm. Then, we inoculated 500 mL LB broth with 1/100 dilutions of the culture and grew the cells at 37°C with shaking at 150 rpm until the optical density at 600 nm (OD600) reached 1.0. We then removed the bacterial cells by centrifugation at 6,000

min and filtered the resulting supernatant hollow-fiber membrane (GE Healthcare)using

Benchtop System (GE Healthcare, Chicago, Illinois, USA) We then concentrated the samples by ultrafiltration through a 100kDa hollow-fiber membrane (GE Healthcare) using the QuixStand Benchtop System. We centrifuged the resulting filtrate again using a 0.45μm vacuum filter and further purified the collected OMVs by ultracentrifugation at 150,000

at 4°C. We then resuspended the purified OMVs in phosphate buffered saline (PBS). We determined the protein concentration using the Bradford assay (Bio

California, USA). Finally, we checked the sterility of the purified OMVs using blood agar plates and then stored them at -80°C.

Transmission electron microscopy (TEM) analysis

To prepare TEM samples, we pelleted overnight cultur 6,000g for 10 min, fixed them with 2.5% glutaraldehyde for 2 h, and washed them. We post-fixed the samples in 1% osmium tetroxide for 1 h, dehydrated them in a graded ethanol series, and embedded them in epoxy resin in beam capsules. We then prepared thin sections using diamond knives on an MTX microtome (RMC Boeckeler Instruments, Tucson, AZ, USA) and stained them with 3% uranyl acetate and lead citrate. For the OMV samples, we applied the purified OMVs to copper grids (Electron Microscopy Sciences) and stained them with 2% uranyl acetate. We visualized the samples

TEM (Hitachi, Japan) operated at 120 kV. SDS-PAGE

We resuspended proteins from whole-cell lysates, culture supernatants, and purified OMV fractions in SDS sample buffer and boiled them for 5 min. We then subjected 10

International Journal Of Current Medical And Pharmaceutical Research, Vol. 4, Issue, 3(A),

pp.3171-is not directly associated with bacterial adherence to epithelial toxic extracellular complexes from K. has been shown to be responsible for causing damage to lung tissues and to be related to K. pneumoniae We speculate that the toxic extracellular may be OMVs that

The OMVs produced by bacteria during normal growth are essential for survival and pathogenesis during host infection16. Recent studies have revealed some of the diverse functions of OMVs; however, the mechanisms by which OMVs are formed and acquire specific proteins, as well as the ultimate functions of the OMVs, remain to be elucidated. In this study, we investigated the production and proteome composition of

ATCC 13883.

ATCC 13883 from the American Type Culture Collection. We cultured the cells in Bertani (LB) medium (Difco, Leeuwarden, Netherlands) at 37°C. We purified OMVs from supernatants of bacterial . We transferred a single ATCC 13883 to 5 mL LB broth (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0) and incubated the culture for 8 hat 37°Cwith shaking at 150 rpm. Then, we inoculated 500 mL LB broth with 1/100 dilutions of the culture ith shaking at 150 rpm until the ) reached 1.0. We then removed the bacterial cells by centrifugation at 6,000g for 15 through a 0.2μm using the QuixStand Benchtop System (GE Healthcare, Chicago, Illinois, USA). by ultrafiltration through a fiber membrane (GE Healthcare) using the We centrifuged the resulting vacuum filter and further purified the collected OMVs by ultracentrifugation at 150,000g for 3 h We then resuspended the purified OMVs in phosphate-buffered saline (PBS). We determined the protein

assay (Bio-Rad, Berkeley, California, USA). Finally, we checked the sterility of the purified OMVs using blood agar plates and then stored them at

Transmission electron microscopy (TEM) analysis

To prepare TEM samples, we pelleted overnight cultures at for 10 min, fixed them with 2.5% glutaraldehyde for 2 fixed the samples in 1% osmium , dehydrated them in a graded ethanol series, and embedded them in epoxy resin in beam capsules. We then thin sections using diamond knives on an MTX microtome (RMC Boeckeler Instruments, Tucson, AZ, USA) and stained them with 3% uranyl acetate and lead citrate. For the OMV samples, we applied the purified OMVs to copper and stained them with 2% uranyl acetate. We visualized the samples with anH-7500

cell lysates, culture supernatants, and purified OMV fractions in SDS-PAGE r and boiled them for 5 min. We then subjected 10

μg of each purified protein sample to electrophoresis on a 12% Tris-glycine gel and subsequently stained the gel with CBB R 250.

Identification of proteins associated with the OMVs

We performed one-dimen

chromatography-tandem mass spectrometry (1

MS/MS) to characterize the proteins in the OMVs. We separated the proteins by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS

them within the gel. We resolved the protein digests in 15μl 0.02% formic acid and 0.5% acetic acid and concentrated them using an MGU30-C18 trapping column (LC Packings) and a nano-column (C18 Reverse-Phase Column, Proxeon). We then eluted the samples by 0–65% acetoni

acquired all MS and MS/MS spectra using an LCQ ion trap mass spectrometer in data

identify the proteins, we compared

K. pneumoniae ATCC 13383 genomic data from NCBInr and the decoy sequence database, allowing for the oxidation of methionine, the carbamidomethylation of cysteines, a single missed trypsin cleavage, a peptide tolerance of 1.5 Da, and a fragment mass tolerance of 1.5 Da.

RESULTS AND DISCUSSION

Purification and characterization of OMVs from K. pneumoniae ATCC 13883

Gram-negative and Gram-positive bacteria during normal growth3. OMVs

communities, enabling intercellular signaling that facilitates predation or activates virulence factors in response to quorum sensing1.

We examined the production and secretion of ATCC 13883 OMVs during in vitro

thin sections of K. pneumoniae

reveal anyevidence of OMV secretion on the bacterial outer membrane (Fig. 1A). To confirm the presence of secretory OMVs in the bacterial culture supernatants, we removed the bacterial cells and applied previously described purifi methods9. TEM analysis of the purified supernatants revealed spherical, bilayered vesicles

nmin diameter (Fig. 1B).

A

Figure 1 Electron micrographs of Klebsiella pneumoniae

(A) Thin-section TEM image of Klebsiella pneumoniae

Negative-stained TEM of purified OMVs, showing their homogeneous sizes of 30–40 nm.

Most of the vesicles had diameters of 4

2A). A previous analysis of native OMV proteins by SDS PAGE demonstrated that many of the proteins were similar in size to outer-membrane, periplasmic, and intracellular

-3176, March, 2018

μg of each purified protein sample to electrophoresis on a 12% glycine gel and subsequently stained the gel with CBB

R-Identification of proteins associated with the OMVs

dimensional electrophoresis-liquid tandem mass spectrometry (1-DE-LC-MS/MS) to characterize the proteins in the OMVs. We separated the proteins by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and digested

he gel. We resolved the protein digests in 15μl 0.02% formic acid and 0.5% acetic acid and concentrated them C18 trapping column (LC Packings) and a Phase Column, Proxeon). We then 65% acetonitrile for 80 min. We acquired all MS and MS/MS spectra using an LCQ-Deca ESI ion trap mass spectrometer in data-dependent mode. To identify the proteins, we compared the MS/MS spectra to the ATCC 13383 genomic data from NCBInr and equence database, allowing for the oxidation of methionine, the carbamidomethylation of cysteines, a single missed trypsin cleavage, a peptide tolerance of 1.5 Da, and a fragment mass tolerance of 1.5 Da.

RESULTS AND DISCUSSION

characterization of OMVs from K.

positive bacteria secrete OMVs are important parts of microbial communities, enabling intercellular signaling that facilitates virulence factors in response to quorum

We examined the production and secretion of K. pneumoniae in vitro culture. TEM analysis of K. pneumoniae ATCC 13883 cells did not reveal anyevidence of OMV secretion on the bacterial

outer-). To confirm the presence of secretory OMVs in the bacterial culture supernatants, we removed the bacterial cells and applied previously described purification TEM analysis of the purified supernatants revealed spherical, bilayered vesicles approximately 30 mm to 100

B

Klebsiella pneumoniae ATCC 13883 cells.

Klebsiella pneumoniae ATCC 13883. (B)

stained TEM of purified OMVs, showing their homogeneous sizes of 40 nm.

Most of the vesicles had diameters of 40 mm to 60 nm (Fig. A previous analysis of native OMV proteins by SDS-PAGE demonstrated that many of the proteins were similar in

(3)

International Journal Of Current Medical And Pharmaceutical Research

(Fig. 2B). Although the protein bands of the culture supernatant and the OMVs were highly similar, the protein profiles differed among the bacterial-cell lysates, culture supernatant, and OMVs. Those results suggest that particular proteins are sorted into the OMVs secreted by

A

Figure 2 Size distributions and protein profiles of native OMVs. (A) The

diameters of 42 native OMVs were calculated from six separate TEM images. A major fraction of the vesicles (60%) were 40–60 nm in diameter. (B) CBB stained SDS-PAGE of OMVs, whole-cell lysate (WC), and molecular weight

maker (M, kDa).

LC-MS/MS analysis of the OMV fraction and the subcellular distribution of the identified proteins

LC-MS/MS analysis identified 159 unique proteins in the secreted K. pneumoniae OMVs (Table S1). OmpX, OmpA, LamB2, and TolB were among the most abundant proteins in the OMVs, whereas other proteins, such as TraU, CysG1, TdcD, and YcfS, were less abundant17. The 159 proteins that we identified have not been identified previously in pneumoniae OMV studies, suggesting that specific proteins may be sorted into the OMVs. Of the 159 proteins, 24 (15.0%), 13 (8.2%), 29 (18.2%), 13 (8.2%), and 80 (50.3%) were derived from the outer-membrane, periplasm, inner membrane, extracellular space, and cytoplasm,

(Fig. 3A). The results of our comprehensive proteome analysis suggest that many of the proteins in the K. pneumoniae originate from the bacterial outer-membrane.

The OMVs contained some major vesicular proteins that are major components of the bacterial outer-membrane, including OmpA, OmpC, OmpX, and YciD9. They also contained some less abundant outer-membrane proteins, including BtuB, FhuA, FepA, LamB2, PldA, and YbiL, and periplasmic proteins, including DegP, DegQ, HlpA, TraU, and TolB. They did not, however, contain the periplasmic proteins that had the highest levels of expression in previous reports, such as LivJ, HdeA, and OppA18. The OMVs also contained some inner membrane proteins; including ChaA, Cmr, CopA, and HflB; extracellular proteins; including NlpD, YibP, YgdR, and YgeR; and ribosomal and metabolic proteins.A number of cytoplasmic proteins that were previously found in OMVs, such as GapA, GltA, GlyA, and SdhC19, did not appear in the K. pneumoniae ATCC 13883 OMVs. Our results indicate that the protein composition of OMVs is not strictly related to the abundance of the proteins within the cell.

results suggest the existence of specific mechanisms that sort proteins into OMVs at certain sites with distinct protein compositions, although further study will be necessary to confirm that hypothesis.

OMVs were once thought to lack inner cytoplasmic components. OMVs from E. coli enterotoxigenic E. coli (ETEC), and P. aeruginosa

International Journal Of Current Medical And Pharmaceutical Research, Vol. 4, Issue, 3(A),

pp.3171-Although the protein bands of the culture rnatant and the OMVs were highly similar, the protein cell lysates, culture Those results suggest that particular into the OMVs secreted by K. pneumoniae.

B

Size distributions and protein profiles of native OMVs. (A) The diameters of 42 native OMVs were calculated from six separate TEM images.

60 nm in diameter. (B) CBB-cell lysate (WC), and molecular weight

MS/MS analysis of the OMV fraction and the subcellular

159 unique proteins in the ). OmpX, OmpA, were among the most abundant proteins in the OMVs, whereas other proteins, such as TraU, CysG1, . The 159 proteins that we identified have not been identified previously in K. OMV studies, suggesting that specific proteins may be sorted into the OMVs. Of the 159 proteins, 24 (15.0%), 13 (8.2%), 29 (18.2%), 13 (8.2%), and 80 (50.3%) membrane, periplasm, inner-membrane, extracellular space, and cytoplasm, respectively

comprehensive proteome analysis K. pneumoniae OMVs membrane.

The OMVs contained some major vesicular proteins that are membrane, including . They also contained some membrane proteins, including BtuB, , LamB2, PldA, and YbiL, and periplasmic proteins, including DegP, DegQ, HlpA, TraU, and TolB. They the periplasmic proteins that had the highest levels of expression in previous reports, such as LivJ, lso contained some inner-membrane proteins; including ChaA, Cmr, CopA, and HflB; extracellular proteins; including NlpD, YibP, YgdR, and YgeR; and ribosomal and metabolic proteins.A number of cytoplasmic proteins that were previously found in OMVs, , did not appear in the . Our results indicate that the protein composition of OMVs is not strictly related to the Furthermore, our existence of specific mechanisms that sort proteins into OMVs at certain sites with distinct protein compositions, although further study will be necessary to

OMVs were once thought to lack inner-membrane and E. coli O157:H7, P. aeruginosa were found

to contain many proteins that are similar in molecular size to cytoplasmic proteins20, however, which is similar to our (Fig. 2B). Cytoplasmic proteins have

OMVs from N. meningitidis mutant

that the presence of cytoplasmic proteins in our OMVs was the result of contamination, our results suggest that transcription factors and ribosomal proteins may be s

OMVs, because vesicles are known to carry DNA and RNA, and outer-membrane proteins may be translated

proximity to their site of integration into the membrane Future studies should seek to determine

are actually components of native OMVs.

Functional classifications of the identified proteins

Of the 159 proteins that we identified, most were involved in the cellular process (30%), information storage and processing (26%), and metabolism (19%; Fig. 3B).

the identified proteins had uncharacterized functions.

the proteins that we identified appeared to function in OMV biogenesis; the elimination of toxins, phages, and competitors; the intercellular transfer of proteins and genet

targeting of host cells; and the modulation of host defense mechanisms22.

A

B

Figure 3 Subcellular locations (A) and COG functional classes (B) of the

pneumoniae ATCC 13883 vesicular proteins.

The mechanism by which OMVs are

Vesicles may by produced when the integrity of the cell envelope is compromised by turgor pressure resulting from the disproportionate expansion of the outer

the peptidoglycan layer or from the periplasmic accum of peptidoglycan fragments23. Among the 159 proteins that we identified, those that were predominant, measuring greater than 0.5 Mol%, included 7 outer

7 periplasmic proteins (21.8%), 1 extracellular protein (3.1%), and 17 cytoplasmic proteins (53.

the predominant proteins were involved in cell

Cytoplasmic 51% Extracellular proteins 8% Metabolism 19% Poorly characterized 25%

-3176, March, 2018

to contain many proteins that are similar in molecular size to , however, which is similar to our results Cytoplasmic proteins have also been found in native mutant20. Although it is possible that the presence of cytoplasmic proteins in our OMVs was the result of contamination, our results suggest that some transcription factors and ribosomal proteins may be sorted into OMVs, because vesicles are known to carry DNA and RNA, may be translated in very close proximity to their site of integration into the membrane21. Future studies should seek to determine if cytoplasmic proteins

tually components of native OMVs.

Functional classifications of the identified proteins

Of the 159 proteins that we identified, most were involved in the cellular process (30%), information storage and processing %; Fig. 3B). Thirty-nine (25%) of the identified proteins had uncharacterized functions. Many of the proteins that we identified appeared to function in OMV biogenesis; the elimination of toxins, phages, and competitors; the intercellular transfer of proteins and genetic material; the targeting of host cells; and the modulation of host defense

A

B

Subcellular locations (A) and COG functional classes (B) of the K.

ATCC 13883 vesicular proteins.

The mechanism by which OMVs are formedis not yet known. Vesicles may by produced when the integrity of the cell envelope is compromised by turgor pressure resulting from the disproportionate expansion of the outer-membrane relative to layer or from the periplasmic accumulation . Among the 159 proteins that we identified, those that were predominant, measuring greater than 0.5 Mol%, included 7 outer-membrane proteins (21.8%), 7 periplasmic proteins (21.8%), 1 extracellular protein (3.1%), 7 cytoplasmic proteins (53.1%; Table 1). A number of the predominant proteins were involved in cell-wall and

(4)

International Journal Of Current Medical And Pharmaceutical Research, Vol. 4, Issue, 3(A), pp.3171-3176, March, 2018

membrane biogenesis, including OmpA, OmpC, YciD, OmpX, Lpp, Tol-Pal, YbgF, and YfgL (Table 1). We also identified themurein hydrolases MltA, MltB, MipA, MltE, Lpp, and EmtA. Murein hydrolases are associated with cell-wall degradation, and their excision may result in turgor pressure due to the accumulation of peptidogly can fragments in the periplasm24. Recent evidence suggests that OMVs are produced regardless of the stability of the bacterial outer-membrane, however, and studies of genetic mutations of lipoproteins have produced controversial results. Therefore, the mechanism underlying OMV biogenesis remains unclear, and further studies are necessary.

E. coli-derived OMVs have been shown to contain organic solvent tolerance proteins (OstA), drug- efflux pumps (TolC), and phage receptors (FepA, FhuA, and OmpA), which may enhance bacterial survival by helping to eliminate toxins and phages25. Proteins that function in the transport of maltose and maltodextrins (LamB), nucleosides (Tsx), vitamin B12 (BtuB), fatty acids (FadL), and ferric iron (FepA, FhuA, YbiL, and Fiu) have also been identified in OMVs26. The functional

significance of those transport proteins in OMVs is still unclear. Some OMVs appear to be predatory in nature, mediating the lysis of microbial competitors. For example, E. coli and P. aeruginosa secrete OMVs containing murein hydrolases that may confer a competitive advantage by killing other bacteria22. We identified several murein hydrolases in the K. pneumonia OMVs, and it is possible that the presence of those proteins in secreted OMVs helps to eliminate competitors by lysing the membranes of other bacteria.

The K. pneumoniae OMVs contained a number of cytoplasmic proteins that are known to be associated with the outer-membrane, including ribosomal proteins (Rps and Rpl families), tRNA synthetases (GlyA and GlyS), and elongation factors (TufA, Tsf, and FusA)27. We also identified several metabolic enzymes (AceF, FadJ, and CysG1). Considering previous reports, our results suggest that K. pneumoniae OMVs may facilitate the transfer of DNA and RNA, as well as Table 1 Major proteins identified from Klebsiella pneumoniae ATCC 13883-derived native OMVs (Mol%>0.5%)

Gene Protein

accession Protein name

Protein length

MW

(Da)a) emPAI Mol%

Protein

scoreb)

Protein

matchesc)

Outer-membrane

1 OmpX A6T6Q8 Outer-membrane protein X 170 18463 5.95 0.030 1290 54

2 OmpA A6T751 Outer-membrane protein 3a 356 38044 5.78 0.029 1260 67

3 LamB2 A6TGU6 Maltoporin 2 429 47776 1.34 0.007 389 13

4 OmpC A6TBT2 Outer-membrane pore protein

1b 367 40095 1.75 0.009 354 17

5 MipA A6T7Q6

Scaffolding protein for murein-synthesizing

holoenzyme

248 28068 3.19 0.016 341 15

6 YraP A6TEG9 Putative periplasmic protein 191 20052 2.28 0.011 277 8

10 YciD A6T7W0 Colicin S4 receptor; putative

transport protein 224 24336 1.28 0.006 188 7

Periplasm

1 TolB A6T6G6 Translocation protein TolB 418 44549 1.99 0.010 383 18

2 Pal A6T6G7 Peptidoglycan-associated

lipoprotein 174 18863 7.21 0.036 358 24

3 Lpp A6TAE1 Murein lipoprotein 78 8386 8.5 0.042 316 16

4 YbgF A6T6G8 Putative periplasmic protein 264 28201 1.71 0.009 283 9

8 EmtA A6TAW0

Endo-type membrane-bound lytic murein transglycosylase

A

203 22358 1.93 0.010 169 6

10 YajG A6T5H7 Putative lipoprotein 181 19580 1.26 0.006 127 6

12 RcsF A6T4Z9 Regulator in colanic acid

synthesis; interacts with RcsB 135 14316 1.28 0.006 119 4

Extracellular proteins

1 YgdR A6TDG9 Putative uncharacterized

protein ygdR 72 7913 16.74 0.083 319 19

Cytoplasm

1 RpsE A6TEV5 30S ribosomal protein S5 167 17647 3.81 0.019 311 11

3 RpsB A6T4X1 30S ribosomal protein S2 241 26699 4.21 0.021 299 25

5 RplE A6TEW0 50S ribosomal protein L5 179 20301 4.84 0.024 248 12

6 RplV A6TEW7 50S ribosomal protein L22 110 12196 16.63 0.083 228 10

8 RplI A6THB4 50S ribosomal protein L9 149 15793 1.72 0.009 221 8

9 RplJ A6TGN8 50S ribosomal protein L10 165 17800 1.44 0.007 215 9

10 RplO A6TEV3 50S ribosomal protein L15 144 15134 1.84 0.009 194 7

11 RplF A6TEV7 50S ribosomal protein L6 177 18844 1.86 0.009 184 5

13 RpsM A6TEV1 30S ribosomal protein S13 118 13210 1.43 0.007 156 6

14 RpsF A6THB1 30S ribosomal protein S6 131 15117 1.84 0.009 143 5

16 RpmB A6TFM8 50S ribosomal protein L28 78 9006 1.34 0.007 119 4

21 RpmC A6TEW4 50S ribosomal protein L29 63 7243 1.83 0.009 106 2

23 RplP A6TEW5 50S ribosomal protein L16 136 15250 1.17 0.006 104 3

26 RpsR A6THB3 30S ribosomal protein S18 75 8986 1.34 0.007 86 5

34 RpsP A6TCL7 30S ribosomal protein S16 82 9090 1.32 0.007 73 3

36 EcnA A6TH66 Entericidin A 43 4434 1.22 0.006 67 1

40 RpmD A6TEV4 50S ribosomal protein L30 59 6514 2.11 0.011 60 2

(5)

International Journal Of Current Medical And Pharmaceutical Research, Vol. 4, Issue, 3(A), pp.3171-3176, March, 2018

that of metabolic enzymes and proteins involved in translation, to other Enterobacteriaceae.

Many Gram-negative bacteria, including ETEH, enterohemorrhagic E. coli, Neisseria meningitidis, and P. aeruginosa, increase their virulence by delivering toxin-containing vesiclesto host cells2. OMV components synergistically modulate the host immune response by interacting with neighboring epithelial cells and immune cells. The most abundant immunogenic constituent of OMVs is LPS. OMVs extracted from pathogenic bacteria such as N. meningitidis are now being used in clinical trials in an effort to exploit their immune-stimulating effects22. The K. pneumoniae OMVs contained outer-membrane proteins and porins, including OmpA, OmpC, YciD, and OmpX, that have been associated with immune responses and the induction of leukocyte chemotaxis22. OmpA enhances LPS uptake by macrophages and contributes to the invasion of endothelial cells in the microvasculature of the brain28. OMVs also contain immune-activating PAMPs, including porins, flagellins, and peptidoglycans20, which interact with host cells and promote inflammation in a number of Gram-negative pathogens3,22.

CONCLUSION

We showed that K. pneumoniae ATCC 13883 produced and secreted OMVs during in vitro culture. We used LC-MS/MS analysis to identify a total of 159 K. pneumoniae vesicular proteins, including 24 outer-membrane proteins (15.0%), 13 periplasmic proteins (8.2%), 29 inner-membrane proteins (18.2%), 13 extracellular proteins (8.2%), and 80 cytoplasmic proteins (50.3%). In agreement with previous reports, the subcellular distribution of the proteins revealed that theK. pneumoniae OMVs were rich in outer-membrane proteins and sparse in inner-membrane proteins 21. The OMVs also contained many vesicular proteins that function in the exclusion of phages, toxins, and competitors, the intercellular transfer of genetic material and proteins, the targeting of host cells, and the modulation of host defenses. The genetics and pathogenesis of the opportunistic pathogen K. pneumonia have been well established in the literature. Our study provides information that will help to create a more detailed and informed picture of the OMVs produced by Gram-negative bacteria, including the underlying mechanisms of OMV biogenesis and the activities of OMVs in multi-drug resistance. A greater understanding of the roles that OMVs play in bacterial lifecycles may lead to new approaches to combat bacterial pathogenesis.

Acknowledgements

The Basic Science Research Program, through the National Research Foundation of Korea (NRF), funded by the Ministry of Science ICT & Future Planning (NRF-2014R1A1A1002349) and the 2014 Eulji University Research Grant gave support to this research.

References

1. Kadurugamuwa, J. L., Beveridge, T. J.,Bacteriolytic effect of membrane vesicles from Pseudomonas aeruginosa on other bacteria including pathogens: conceptually new antibiotics. J. Bacteriol. 1996, 178, 2767-2774.

2. Kuehn, M. J., Kesty, N. C., Bacterial outer membrane vesicles and the host-pathogen interaction. Genes. Dev. 2005, 19, 2645-2655.

3. Kulp, A., Kuehn, M. J., Biological functions and biogenesis of secreted bacterial outer membranevesicles. Annu. Rev. Microbiol.2010,64, 163-184.

4. Nikaido, H., Isolation of outer membrane. Methods. Enzymol. 1994, 235, 225-234.

5. Lindmark, B., Rompikuntal, P. K., Vaitkevicius, K., Song, T., Mizunoe, Y., Uhlin, B. E., Guerry, P., Wai, S. N.,Outer membrane vesicle-mediated release of cytolethal distending toxin (CDT) from Campylobacter jejuni.BMC. Microbiol.2009, 9, 220.

6. Kesty, N. C., Mason, K. M., Reedy, M., Miller, S. E., Kuehn, M. J.,Enterotoxigenic Escherichia coli vesicles target toxin delivery into mammalian cells. EMBO. J.2004, 23, 4538-4549.

7. Bomberger, J. M., Maceachran, D. P., Coutermarsh, B. A., Ye, S., O'Toole, G. A., Stanton, B. A.,Long-distance delivery of bacterial virulence factors by Pseudomonas aeruginosa outer membrane vesicles.PLoS. Pathog.2009, 5, e1000382.

8. Keenan, J., Day, T., Neal, S., Cook, B., Perez-Perez, G., Allardyce, R., Bagshaw, P., A role for the bacterial outer membrane in the pathogenesis of Helicobacter pylori infection. FEMS Microbiol Lett.2000, 182, 259-264.

9. Wai, S. N., Lindmark, B., Soderblom, T., Takade, A., Westermark, M., Oscarsson, J., Jass, J., Richter-Dahlfors, A., Mizunoe, Y., Uhlin, B.,Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin. Cell. 2003, 115, 25-35. 10. Keynan, Y., Rubinstein, E., The changing face of

Klebsiella pneumoniae infections in the community. Int. J. Antimicrob. Agents.2007, 30, 385-389.

11. Cortés, G., Borrell, N., de Astorza, B., Gómez, C., Sauleda, J., Albertí, S., Molecular analysis of the contribution of the capsular polysaccharide and the lipopolysaccharide O side chain to the virulence of Klebsiella pneumoniae in a murine model of pneumonia. Infect. Immun.2002, 70, 2583-2590. 12. Lawlor, M. S., Hsu, J., Rick, P. D., Miller, V. L.,

Identification of Klebsiella pneumoniaevirulence determinants using an intranasal infection model. Mol. Microbiol.2005, 58, 1054-1073.

13. Nassif, X., Sansonetti, P. J., Correlation of the virulence of Klebsiella pneumoniae K1 and K2 with the presence of a plasmid encoding aerobactin. Infect. Immun.1986, 54, 603-608.

14. Straus, D. C., Production of an extracellular toxic complex by various strains of Klebsiella pneumoniae. Infect. Immun.1987, 55, 44-48.

15. Lee JC, Lee EJ, Lee JH, Jun SH, Choi CW, Kim SI, Kang SS, Hyun S, Klebsiella pneumoniae secretes outer membrane vesicles that induce the innate immune response. FEMS microbiology letters. 2012, 331: 17-24. 16. Henry, T., Pommier, S., Journet, L., Bernadac, A., grovel, J. P., Lloubes, R., Improved methods for producing outer membrane vesicles in Gram-negative bacteria. Res. Microbiol. 2004, 155, 437-446.

(6)

International Journal Of Current Medical And Pharmaceutical Research, Vol. 4, Issue, 3(A), pp.3171-3176, March, 2018

18. Ratajczak, j., Wysoczynski, M., Hayek, F., Janowska-Wieczorek, A., Ratajczak, M. Z., Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication. Leukemia 2006, 20, 1487-1495.

19. Buttner, K., Bernhardt, J., Scharf, C., Schmid, R., Mader, U., Eymann, C., Antelmann, H., Volker, A., Volker, U., Hecker, M., A comprehensive two-dimensional map of cytosolic proteins of Bacillus subtilis. Electrophoresis 2001, 22, 2908-2935.

20. Bauman, S. J., Kuehn, M. J., Purification of outer membrane vesicles from Pseudomonas aeruginosa and their activation of an IL-8 response. Microbes Infect. 2006, 8, 2400-2408.

21. Dorward, D. W., Garon, C. F., Judd, R. C., Export and intercellular transfer of DNA via membrane blebs of Neisseria gonorrhoeae. J. Bacteriol. 1989, 171, 2499-2505.

22. Galdiero, M., Folgore, A., Molitierno, M., Greco, R., Porins and lipopolysaccharide (LPS) from Salmonella typhimurium induce leucocytetransmigration through human endothelial cells in vitro. Clin. Exp. Immunol.1999, 116:453-461.

23. Zhou, L., Srisatjaluk, R., Justus, D. E., Doyle, R, On the origin of membrane vesicles in gram-negative bacteria. J., FEMS Microbiol. Lett. 1998, 163, 223-228.

24. Lommatzsch, J., Templin, M. F., Kraft, A. R., Vollmer, W., Holtje, J. V., Outer membrane localization of murein hydrolases : MltA, a third lipoprotein lytic transglycosylase in Escherichia. coli. J. Bacteriol. 1997, 179, 5465-5470.

25. Kobayashi, H., Uematsu, K., Hirayama, H., Horikoshi, K., Novel toluene elimination system in a toluene-tolerant microorganism. J. Bacteriol. 2000, 182, 6451-6455.

26. Molloy, M. P., Herbert, B. R., slade, M. B., Rabilloud, T., Nouwens, A. S., Williams, K. L., Gooley, A. A., Proteomic analysis of the Escherichia coli outer membrane. Eur. J. Biochem. 2000, 267, 2871-2881. 27. Vipond, C., Suker, J., Jones, C., Tang, C., Feavers, I.

M., Wheeler, J. X., Proteomic analysis of a meningococcal outer membrane vesicle vaccine prepared from the group B strain NZ98/254. Proteomics 2006, 6, 3400-3413.

28. Prasadarao, N. V., Wass, C. A., Weiser, J. N., Stins, M. F. Huang, s. H., Kim, K. S., Outer membrane protein A of Escherichia coli contributes to invasion of brain microvascular endothelial cells. Infect. Immun. 1996, 64, 146-153.

How to cite this article:

Hee Sang You et al (2018) 'Characteristics of Outer-Membrane Vesiclesderived From Klebsiella Pneumoniae Atcc 13883', International Journal of Current Medical And Pharmaceutical Research, 04(3), pp. 3171-3176.

Figure

Figure 1 ATCC 13883. (B) stained TEM of purified OMVs, showing their homogeneous sizes of Klebsiella pneumoniae Klebsiella pneumoniae Klebsiella pneumoniae ATCC 13883 cells
Figure 2 Size distributions and protein profiles of native OMVs. (A) The Size distributions and protein profiles of native OMVs

References

Related documents

7 7 Ultimately, then, from a perspective of American legal practice, Jewish legal and ethical teachings present a model for a consideration of the way religious

This study was part of a larger study to describe how master’s of occupational therapy (MOT) students define and perceive their own creative thinking across the academic program..

Depositional sequences in turbidite successions of the lower Kazusa Group, the Plio-Pleistocene forearc basin fill in the Boso Peninsula, Japan. Spectral Analysis

As in solitary giant cell tumor, the most aggressive behavior of the vast majority of multicentric giant cell tumors is local recurrence, especially in multicentric metachronous GCT

The issues in human resource development and preservation which are examined pragmatically are education, skill development, skill-improvement, training for

Designing friendly environment for disabled people and elderly has been introduced for decades especially in developed countries such as accessible design, barrier

Yield and yield components were maximum in wheat monoculture plots while yield and yield components were minimum in treatments of 10 wheat plants with 90 wild oat

The initial results of the cell problems for the calculation of the dielectric response function compare well with the results that were obtained in [3], where a probabilistic