Je Chul Lee

Outer membrane protein 38 of Acinetobacter baumannii localizes to the mitochondria and induces apoptosis of epithelial cells

Acinetobacter baumannii is an important opportunistic pathogen responsible for nosocomial infection. Despite considerable clinical and epidemiological data regarding the role of A. baumannii in nosocomial infection, the specific virulence factor or pathogenic mechanism of this organism has yet to be elucidated. This study investigated the molecular mechanism of apoptosis on the infection of human laryngeal epithelial HEp‐2 cells with A. baumannii and examined the contribution of outer membrane protein 38 (Omp38) on the ability of A. baumannii to induce apoptosis of epithelial cells. A. baumannii induced apoptosis of HEp‐2 cells through cell surface death receptors and mitochondrial disintegration. The Omp38‐deficient mutant was not as able to induce apoptosis as the wild‐type A. baumannii strain. Purified Omp38 entered the cells and was localized to the mitochondria, which led to a release of proapoptotic molecules such as cytochrome c and apoptosis‐inducing factor (AIF). The activation of caspase‐3, which is activated by caspase‐9, degraded DNA approximately 180 bp in size, which resulted in the appearance of a characteristic DNA ladder. AIF degraded chromosomal DNA approximately 50 kb in size, which resulted in large‐scale DNA fragmentation. These results demonstrate that Omp38 may act as a potential virulence factor to induce apoptosis of epithelial cells in the early stage of A. baumannii infection.

Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells

BackgroundAcinetobacter baumannii is a nosocomial pathogen of increasing importance, but the pathogenic mechanism of this microorganism has not been fully explored. This study investigated the potential of A. baumannii to invade epithelial cells and determined the role of A. baumannii outer membrane protein A (AbOmpA) in interactions with epithelial cells.ResultsA. baumannii invaded epithelial cells by a zipper-like mechanism, which is associated with microfilament- and microtubule-dependent uptake mechanisms. Internalized bacteria were located in the membrane-bound vacuoles. Pretreatment of recombinant AbOmpA significantly inhibited the adherence to and invasion of A. baumannii in epithelial cells. Cell invasion of isogenic AbOmpA- mutant significantly decreased as compared with wild-type bacteria. In a murine pneumonia model, wild-type bacteria exhibited a severe lung pathology and induced a high bacterial burden in blood, whereas AbOmpA- mutant was rarely detected in blood.ConclusionA. baumannii adheres to and invades epithelial cells. AbOmpA plays a major role in the interactions with epithelial cells. These findings contribute to the understanding of A. baumannii pathogenesis in the early stage of bacterial infection.

Proteome analysis of outer membrane vesicles from a clinical Acinetobacter baumannii isolate

The secretion of outer membrane vesicles (OMVs) is one of the major mechanisms by which Gram-negative bacteria deliver effector molecules to host cells. Acinetobacter baumannii is an important opportunistic pathogen in hospital-acquired infections, but the secretion system for effector molecules to induce host cell damage has not been characterized. In the present study, we investigated the secretion of OMVs from a clinical A. baumannii isolate and analyzed the comprehensive proteome of A. baumannii-derived OMVs. Acinetobacter baumannii secreted OMVs into the extracellular milieu during in vitro growth. Using 1-DE and LC-MS/MS protein identification and assignment analysis, 132 different proteins associated with OMVs were identified. These proteins were derived from outer membranes (n=26), periplasmic space (n=6), inner membranes (n=8), cytoplasm (n=43), and unknown localization or multiple localization sites (n=49) according to the cell location prediction programs. Among the proteins associated with OMVs, a potent cytotoxic molecule, outer membrane protein A, was highly enriched and several putative virulence-associated proteins were also identified. These results suggest that OMVs from A. baumannii are an important vehicle designed to deliver effector molecules to host cells.

Acinetobacter baumannii Secretes Cytotoxic Outer Membrane Protein A via Outer Membrane Vesicles

Acinetobacter baumannii is an important nosocomial pathogen that causes a high morbidity and mortality rate in infected patients, but pathogenic mechanisms of this microorganism regarding the secretion and delivery of virulence factors to host cells have not been characterized. Gram-negative bacteria naturally secrete outer membrane vesicles (OMVs) that play a role in the delivery of virulence factors to host cells. A. baumannii has been shown to secrete OMVs when cultured in vitro, but the role of OMVs in A. baumannii pathogenesis is not well elucidated. In the present study, we evaluated the secretion and delivery of virulence factors of A. baumannii to host cells via the OMVs and assessed the cytotoxic activity of outer membrane protein A (AbOmpA) packaged in the OMVs. A. baumannii ATCC 19606T secreted OMVs during in vivo infection as well as in vitro cultures. Potential virulence factors, including AbOmpA and tissue-degrading enzymes, were associated with A. baumannii OMVs. A. baumannii OMVs interacted with lipid rafts in the plasma membranes and then delivered virulence factors to host cells. The OMVs from A. baumannii ATCC 19606T induced apoptosis of host cells, whereas this effect was not detected in the OMVs from the ΔompA mutant, thereby reflecting AbOmpA-dependent host cell death. The N-terminal region of AbOmpA22-170 was responsible for host cell death. In conclusion, the OMV-mediated delivery of virulence factors to host cells may well contribute to pathogenesis during A. baumannii infection.

Changes in Gene Cassettes of Class 1 Integrons among Escherichia coli Isolates from Urine Specimens Collected in Korea during the Last Two Decades

ABSTRACT Gene cassettes of class 1 integrons in Escherichia coli isolates from urine specimens collected in Korea during the last 2 decades were characterized. intI1 was detected in 54% of the isolates, yet gene cassette regions were amplified in only 43% of the isolates. intI2 was detected in 29 (5%) isolates, and no intI3 was detected in this study. Twenty-one different genes, including genes encoding resistance to antibiotics, an alcohol dehydrogenase gene (adhE), and unknown genes, were detected. The genes most commonly found in class 1 integrons were those for aminoglycoside and trimethoprim resistance. The occurrence of aminoglycoside resistance genes in class 1 integrons decreased, and the presence of dfr genes increased rapidly, during the last 2 decades. Single-gene cassettes were predominant during the 1980s, while multigene cassettes predominated from the 1990s on. The aadA1, aadA2, and blaP1-aadA2 gene cassettes were frequently found in isolates from the 1980s but were not detected in isolates recovered since 2000. dfrA12-aadA2 and dfrA17-aadA5 were the most prevalent gene cassettes among isolates recovered from the 1990s on. In conclusion, class 1 integrons would appear to be responsible for resistance to antibiotics commonly used to treat urinary tract infections, and selection of a specific gene cassette was found to occur over the course of time.

Acinetobacter baumannii outer membrane protein A targets the nucleus and induces cytotoxicity

Acinetobacter baumannii is an emerging opportunistic pathogen responsible for healthcare‐associated infections. The outer membrane protein A of A. baumannii (AbOmpA) is the most abundant surface protein that has been associated with the apoptosis of epithelial cells through mitochondrial targeting. The nuclear translocation of AbOmpA and the subsequent pathology on host cells were further investigated. AbOmpA directly binds to eukaryotic cells. AbOmpA translocates to the nucleus by a novel monopartite nuclear localization signal (NLS). The introduction of rAbOmpA into the cells or a transient expression of AbOmpA–EGFP causes the nuclear localization of these proteins, while the fusion proteins of AbOmpAΔNLS–EGFP and AbOmpA with substitutions in residues lysine to alanine in the NLS sequences represent an exclusively cytoplasmic distribution. The nuclear translocation of AbOmpA induces cell death in vitro. Furthermore, the microinjection of rAbOmpA into the nucleus of Xenopus laevis embryos fails to develop normal embryogenesis, thus leading to embryonic death. We propose a novel pathogenic mechanism of A. baumannii regarding the nuclear targeting of the bacterial structural protein AbOmpA.

Staphylococcus aureus produces membrane-derived vesicles that induce host cell death.

Gram-negative bacteria produce outer membrane vesicles that play a role in the delivery of virulence factors to host cells. However, little is known about the membrane-derived vesicles (MVs) produced by Gram-positive bacteria. The present study examined the production of MVs from Staphylococcus aureus and investigated the delivery of MVs to host cells and subsequent cytotoxicity. Four S. aureus strains tested, two type strains and two clinical isolates, produced spherical nanovesicles during in vitro culture. MVs were also produced during in vivo infection of a clinical S. aureus isolate in a mouse pneumonia model. Proteomic analysis showed that 143 different proteins were identified in the S. aureus-derived MVs. S. aureus MVs were interacted with the plasma membrane of host cells via a cholesterol-rich membrane microdomain and then delivered their component protein A to host cells within 30 min. Intact S. aureus MVs induced apoptosis of HEp-2 cells in a dose-dependent manner, whereas lysed MVs neither delivered their component into the cytosol of host cells nor induced cytotoxicity. In conclusion, this study is the first report that S. aureus MVs are an important vehicle for delivery of bacterial effector molecules to host cells.

Serum resistance of Acinetobacter baumannii through the binding of factor H to outer membrane proteins

Bacteremia is a common systemic disease caused by Acinetobacter baumannii, an important hospital-acquired pathogen among critically ill patients. The complement system is central to innate immune defense against invading bacteria in the blood. The present study investigated the susceptibility of clinical A. baumannii isolates to normal human sera (NHS), and determined the resistance mechanism of A. baumannii against complement-mediated lysis. The survival of A. baumannii isolates from bacteremic patients was significantly decreased in undiluted NHS, but they were resistant to 40% NHS. The alternative complement pathway was responsible for the direct killing of bacteria. The main regulator of the alternative complement pathway, factor H, bound to the surface of live A. baumannii treated with NHS. Factor H interacted with the outer membrane proteins with molecular sizes of 38 (AbOmpA), 32, and 24 kDa. The isogenic AbOmpA(-) mutant was highly susceptible to NHS in comparison with the wild-type A. baumannii strain, suggesting that AbOmpA was an important complement regulator-acquiring surface protein. These results indicate that A. baumannii evades complement attack through the acquisition of factor H to their surface.

Mechanism of anchoring of OmpA protein to the cell wall peptidoglycan of the gram-negative bacterial outer membrane

The outer membrane protein A (OmpA) plays important roles in anchoring of the outer membrane to the bacterial cell wall. The C‐terminal periplasmic domain of OmpA (OmpA‐like domain) associates with the peptidoglycan (PGN) layer noncovalently. However, there is a paucity of information on the structural aspects of the mechanism of PGN recognition by OmpA‐like domains. To elucidate this molecular recognition process, we solved the high‐resolution crystal structure of an OmpA‐like domain from Acinetobacter baumannii bound to diaminopimelate (DAP), a unique bacterial amino acid from the PGN. The structure clearly illustrates that two absolutely conserved Asp271 and Arg286 residues are the key to the binding to DAP of PGN. Identification of DAP as the central anchoring site of PGN to OmpA is further supported by isothermal titration calorimetry and a pulldown assay with PGN. An NMR‐based computational model for complexation between the PGN and OmpA emerged, and this model is validated by determining the crystal structure in complex with a synthetic PGN fragment. These structural data provide a detailed glimpse of how the anchoring of OmpA to the cell wall of gram‐negative bacteria takes place in a DAP‐dependent manner.—Park, J. S., Lee, W. C., Yeo, K. J., Ryu, K.‐S., Kumarasiri, M., Hesek, D., Lee, M., Mobashery, S., Song, J. H., Lim, S. I., Lee, J. C., Cheong, C., Jeon, Y. H., Kim, H.‐Y. Mechanism of anchoring of OmpA protein to the cell wall peptidoglycan of the gram‐negative bacterial outer membrane. FASEB J. 26, 219–228 (2012). www.fasebj.org

Prevalence of the ST239 Clone of Methicillin-Resistant Staphylococcus aureus and Differences in Antimicrobial Susceptibilities of ST239 and ST5 Clones Identified in a Korean Hospital

ABSTRACT A total of 188 nonduplicate methicillin-resistant Staphylococcus aureus (MRSA) isolates obtained between 2001 and 2004 in a university hospital in Daegu, Korea, were analyzed for their clonal types by molecular typing techniques, including multilocus sequence typing, spaA typing, staphylococcal chromosomal cassette mec (SCCmec) typing, and pulsed-field gel electrophoresis (PFGE). They were examined for their antimicrobial susceptibilities. The majority (87%) of MRSA isolates belonged to sequence type 239 (ST239; n = 100; 53%) and ST5 (n = 63, 34%) on the basis of sequence typing. MRSA isolates belonging to ST239 were genotypically homogeneous, while those belonging to ST5 showed variations in spaA type, SCCmec type, and PFGE patterns. The rates of resistance of the MRSA isolates belonging to ST239 to trimethoprim, sulfamethoxazole, tobramycin, gentamicin, erythromycin, and tetracycline were significantly higher than those of the isolates belonging to ST5 (P < 0.05). This study demonstrated that the ST239 clone, while rarely detected in Korea, was prevalent and that the antimicrobial susceptibility of the ST239 clone was significantly different from that of the ST5 clone.