<i>Pseudomonas aeruginosa</i> injects NDK into host cells through a type III secretion system

Neeld, Dennis,Jin, Yongxin,Bichsel, Candace,Jia, Jinghua,Guo, Jianhui,Bai, Fang,Wu, Weihui,Ha, Un-Hwan,Terada, Naohiro,Jin, Shouguang Society for General Microbiology 2014 Microbiology Vol.160 No.7

<P><I>Pseudomonas aeruginosa</I> is a Gram-negative opportunistic human pathogen possessing a type III secretion system (T3SS) which injects toxic effector proteins into mammalian host cells. In previous studies, <I>P. aeruginosa</I> strains lacking all of the known type III effectors were shown to cause cytotoxicity upon prolonged infection time. In this study, we report the identification of a new cytotoxin, nucleoside diphosphate kinase (NDK), which is injected into eukaryotic cells in a T3SS-dependent manner. Injection of NDK is inhibited by the presence of previously known effectors of the T3SS, with an effectorless strain injecting the highest amount, suggesting active competition with the known T3SS effectors. NDK is shown to cause a cytotoxic response when expressed in eukaryotic cells, and <I>P. aeruginosa</I> strains harbouring NDK also show a greater toxicity than strains lacking it. Interestingly, the cytotoxic effect of intracellular NDK is independent of its kinase activity. In previous studies, NDK was shown to be secreted into culture supernatants via a type I secretion system and cause cytotoxicity in a kinase-dependent manner. Therefore, the current study highlights an alternative route of NDK secretion as well as two different cytotoxic mechanisms of NDK, depending on the extra- or intra-cellular location of the protein.</P>

<i>Pseudomonas aeruginosa</i> GroEL Stimulates Production of PTX3 by Activating the NF-κB Pathway and Simultaneously Downregulating MicroRNA-9

Shin, Heesung,Jeon, Jisu,Lee, Jung-Hoon,Jin, Shouguang,Ha, Un-Hwan American Society for Microbiology 2017 Infection and immunity Vol.85 No.3

<P>As one of the first lines of host defense, monocytes play important roles in clearing infected microbes. The defensive response is triggered by recognition of diverse microbial moieties, including released factors, which modulate host immune responses to establish a harsh environment for clinically important bacterial pathogens. In this study, we found that the expression of PTX3, a soluble form of pattern recognition receptor, was induced by infection with live Pseudomonas aeruginosa or treatment of cells with its supernatant. P. aeruginosa GroEL, a homolog of heat shock protein 60, was identified as one of the factors responsible for inducing the expression of PTX3 in host cells. GroEL induced PTX3 expression by activating the Toll-like receptor 4 (TLR4)-dependent pathway via nuclear factor-kappa B (NF-kappa B), while simultaneously inhibiting expression of microRNA-9, which targets the PTX3 transcript. Finally, by acting as an opsonin, GroEL-induced PTX3 promoted the association and phagocytosis of Staphylococcus aureus into macrophages. These data suggest that the host defensive environment is supported by the production of PTX3 in response to GroEL, which thus has therapeutic potential for clearance of bacterial infections.</P>

Identification of D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in Corynebacterium glutamicum

Jung-Hoon Lee,Yong-Jae Kim,Hee-Sung Shin,Heung-Shick Lee,Shouguang Jin,Un-Hwan Ha 한국미생물학회 2016 The journal of microbiology Vol.54 No.6

Expression of a putative acyltransferase encoded by NCgl- 0350 of Corynebacterium glutamicum is induced by cell-free culture fluids obtained from stationary-phase growth of both C. glutamicum and Pseudomonas aeruginosa, providing evidence for interspecies communication. Here, we further confirmed that such communication occurs by showing that acyltransferase expression is induced by culture fluid obtained from diverse Gram-negative and -positive bacterial strains, including Escherichia coli, Salmonella Typhimurium, Bacillus subtilis, Staphylococcus aureus, Mycobacterium sp. strain JC1, and Mycobacterium smegmatis. A homologous acyltransferase encoded by PA5238 of P. aeruginosa was also induced by fluids obtained from P. aeruginosa as well as other bacterial strains, as observed for NCgl0350 of C. glutamicum. Because C. glutamicum is difficult to study using molecular approaches, the homologous gene PA5238 of P. aeruginosa was used to identify PA5309 as an upstream regulator of expression. A homologous D-amino acid dehydrogenase encoded by NCgl- 2909 of C. glutamicum was cloned based on amino acid similarity to PA5309, and its role in the regulation of NCgl0350 expression was confirmed. Moreover, NCgl2909 played positive roles in growth of C. glutamicum. Thus, we identified a D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in C. glutamicum.

Bradykinin Receptor의 발현에 미치는 녹농균유래 Nucleoside Diphosphate Kinase 및 Flagellin의 효과

김용재,신희성,하운환,Kim, Yong-Jae,Shin, Hee-Sung,Jin, Shouguang,Ha, Un-Hwan 한국미생물학회 2014 미생물학회지 Vol.50 No.4

Immune defense responses against Pseudomonas aeruginosa infection play an important role in maintaining homeostasis in the human body. Previously, we reported that expression of the bradykinin receptor (BR) is induced in response to P. aeruginosa infection. However, the factors responsible for the induction was uncertain. Here, we found that the type III secretion system (T3SS) is responsible for the induction of BR expression, and nucleoside diphosphate kinase (Ndk), as a novel T3SS effector, mediates the upregulation. The Ndk-mediated expression of BR was not induced by fliC mutant treatment, indicating the involvement of flagellin, one of the well-known pathogen-associated molecular patterns (PAMPs). Taken together, this study demonstrated that Ndk cooperates with flagella in the development of defense responses against P. aeruginosa infection. 병원성 균주인 녹농균(Pseudomonas aeruginosa) 감염에 대응하여 나타나는 면역반응은 인체의 항상성 유지에 중요하다. 선행 연구에서 녹농균의 감염에 대응하여 bradykinin receptor (BR)의 발현이 증가됨을 보고하였지만, 발현유도에 관여하는 녹농균 유래인자에 대해서는 보고한 바가 없었다. 이번 연구에서는 녹농균에 의한 BR의 발현은 Type III secretion system (T3SS)이 관여하지만, 기존에 알려진 T3SS인자가 아닌 nucleoside diphosphate kinase (Ndk)에 의한 것으로 조사되었다. 하지만 pDNNDK를 이용한 transfection 실험 결과, Ndk 만으로는 BR의 발현이 유도되지 않았으며, Ndk와 함께 flagella가 필요함을 발견하였다. 이러한 결과는 기존에 보고된 주요 pathogen-associated molecular patterns (PAMPs)인 flagella와 더불어 감염대응에 관여하는 Ndk를 발굴한 의미가 있으며, 녹농균에 의한 질병기전을 이해하는데 도움을 줄 수 있다.

Autoinduction of a genetic locus encoding putative acyltransferase in Corynebacterium glutamicum

Shin, Hee-Sung,Kim, Yong-Jae,Yoo, In-Hwa,Lee, Heung-Shick,Jin, Shouguang,Ha, Un-Hwan Springer-Verlag 2011 Biotechnology letters Vol.33 No.1

Expression and Efficient Purification of Tag-cleaved Active Recombinant Human Insulin-like Growth Factor-II from Escherichia coli

Hongbo Li,Xiaoyan Hui,Peng Li,Aimin Xu,Shiwu Li,Shouguang Jin,Donghai Wu 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.2

Insulin-like growth factor-II (IGF2) is a growth factor for the control of cell proliferation and apoptosis. To explore the clinical use of human IGF2, an efficient method for production of a large amount of active recombinant hIGF2 is necessary. Human IGF2 cDNA was cloned into pET32 vector where it is under the control of an IPTGinducible T7 promoter. High level soluble thioredoxin (Trx)- hIGF2 fusion protein was produced at room temperature following IPTG induction, amounting up to 20% of the total soluble bacterial proteins. The recombinant TrxhIGF2 fusion protein was purified to an approximate 95% purity using Ni+-NTA affinity chromatography with an overall yield of 120 mg protein per liter of bacterial culture. After cleavage of the Trx fusion fragment by recombinant enterokinase, the tag-free recombinant hIGF2 protein (rhIGF2) was purified by passage through the Ni+-NTA affinity column again. Biological activity of the purified hIGF2 was determined by its ability to support NIH/3T3 cells proliferation and to activate AKT signaling pathways. Our results demonstrate that tag-free active rhIGF2 can easily be obtained for various applications from E. coli using the procedure described in this report.

TatC-dependent translocation of pyoverdine is responsible for the microbial growth suppression

이예지,김용재,Jung-Hoon Lee,유형은,Kiho Lee,Shouguang Jin,하운환 한국미생물학회 2016 The journal of microbiology Vol.54 No.2

Infections are often not caused by a colonization of Pseudomonas aeruginosa alone but by a consortium of other bacteria. Little is known about the impact of P. aeruginosa on the growth of other bacteria upon coinfection. Here, cellree culture supernatants obtained from P. aeruginosa suppressed the growth of a number of bacterial strains such as Corynebacterium glutamicum, Bacillus subtilis, Staphylococcus aureus, and Agrobacterium tumefaciens, but had little effect on the growth of Escherichia coli and Salmonella Typhimurium. The growth suppression effect was obvious when P. aeruginosa was cultivated in M9 minimal media, and the suppression was not due to pyocyanin, a well-known antimicrobial toxin secreted by P. aeruginosa. By performing transposon mutagenesis, PA5070 encoding TatC was identified, and the culture supernatant of its mutant did not suppress the growth. HPLC analysis of supernatants showed that pyoverdine was a secondary metabolite present in culture supernatants of the wild-type strain, but not in those of the PA5070 mutant. Supplementation of FeCl2 as a source of iron compromised the growth suppression effect of supernatants and also recovered biofilm formation of S. aureus, indicating that pyoverdine-mediated iron acquisition is responsible for the growth suppression. Thus, this study provides the action of TatC-dependent pyoverdine translocation for the growth suppression of other bacteria, and it might aid understanding of the impact of P. aeruginosa in the complex community of bacterial species upon coinfection.

MKP1 Regulates the Induction of MCP1 by Streptococcus pneumoniae Pneumolysin in Human Epithelial Cells

Hee-Sung Shin,In-Hwa Yoo,Yong-Jae Kim,Ji-Yun Lee,Shouguang Jin,하운환,김형배 한국분자세포생물학회 2010 Molecules and cells Vol.30 No.3

Epithelial cells act as the first line of host defense against microbes by producing a range of different molecules for clearance. Chemokines facilitate the clearance of invaders through the recruitment of leukocytes. Thus, upregulation of chemokine expression represents an important innate host defense response against invading microbes such as Streptococcus pneumoniae. In this study, we report that the expression of Monocyte Chemotactic Protein 1 (MCP1)was highly induced in response to S. pneumoniae in vitro and in vivo. Among numerous virulence factors, pneumococcal pneumolysin was found to be the major factor responsible for this induction. Furthermore, MCP1 induction was mediated by the p38 mitogen-activated protein kinase (MAPK) whose activation was controlled by MAPK phosphatase 1 (MKP1). Therefore, this study reveals novel roles of pneumolysin in mediating MKP1 expression for the regulation of MCP1 expression in human epithelial cells

Nucleoside Diphosphate Kinase and Flagellin from <i>Pseudomonas aeruginosa</i> Induce Interleukin 1 Expression via the Akt/NF-κB Signaling Pathways

Kim, Yong-Jae,Lee, Jung-Hoon,Lee, Yeji,Jia, Jingyue,Paek, Se-Hwan,Kim, Hyong-Bai,Jin, Shouguang,Ha, Un-Hwan American Society for Microbiology 2014 Infection and immunity Vol.82 No.8

<P>Inflammatory responses are a first line of host defense against a range of invading pathogens, consisting of the release of proinflammatory cytokines followed by attraction of polymorphonuclear neutrophils (PMNs) to the site of inflammation. Among the many virulence factors that contribute to the pathogenesis of infections, nucleoside diphosphate kinase (Ndk) mediates bacterially induced toxicity against eukaryotic cells. However, no study has examined how Ndk affects inflammatory responses. The present study examined the mechanisms by which <I>Pseudomonas aeruginosa</I> activates inflammatory responses upon infection of cells. The results showed that bacterial Ndk, with the aid of an additional bacterial factor, flagellin, induced expression of the proinflammatory cytokines interleukin-1α (IL-1α) and IL-1β. Cytokine induction appeared to be dependent on the kinase activity of Ndk and was mediated via the NF-κB signaling pathway. Notably, Ndk activated the Akt signaling pathway, which acts upstream of NF-κB, as well as caspase-1, which is a key component of inflammasome. Thus, this study demonstrated that <I>P. aeruginosa</I>, through the combined effects of Ndk and flagellin, upregulates the expression of proinflammatory cytokines via the Akt/NF-κB signaling pathways.</P>

Regulatory Role of PopN and Its Interacting Partners in Type III Secretion of Pseudomonas aeruginosa

Yang, Hongjing,Shan, Zhiying,Kim, Jaewha,Wu, Weihui,Lian, Wei,Zeng, Lin,Xing, Laijun,Jin, Shouguang American Society for Microbiology 2007 Journal of Bacteriology Vol.189 No.7

<B>ABSTRACT</B><P>The type III secretion system (T3SS) of <I>Pseudomonas aeruginosa</I> plays a significant role in pathogenesis. We have previously identified type III secretion factor (TSF), which is required for effective secretion of the type III effector molecules, in addition to the low calcium signal. TSF includes many low-affinity high-capacity calcium binding proteins, such as serum albumin and casein. A search for the TSF binding targets on the bacterial outer membrane resulted in identification of PopN, a component of the T3SS that is readily detectable on the bacterial cell surface. PopN specifically interacts with Pcr1, and both <I>popN</I> and <I>pcr1</I> mutants have a constitutive type III secretion phenotype, suggesting that the two proteins form a complex that functions as a T3SS repressor. Further analysis of the <I>popN</I> operon genes resulted in identification of protein-protein interactions between Pcr1 and Pcr4 and between Pcr4 and Pcr3, as well as between PopN and Pcr2 in the presence of PscB. Unlike <I>popN</I> and <I>pcr1</I> mutants, <I>pcr3</I> and <I>pcr4</I> mutants are totally defective in type III secretion, while a <I>pcr2</I> mutant exhibits reduced type III secretion. Interestingly, PopN, Pcr1, Pcr2, and Pcr4 are all secreted in a type III secretion machinery-dependent manner, while Pcr3 is not. These findings imply that these components have important regulatory roles in controlling type III secretion.</P>