Un-Hwan Ha

DsbA of Pseudomonas aeruginosa Is Essential for Multiple Virulence Factors

ABSTRACT DsbA is a periplasmic thiol:disulfide oxidoreductase which contributes to the process of protein folding by catalyzing the formation of disulfide bonds. In this study, we demonstrate that the dsbA gene is required for the expression of the type III secretion system under low-calcium inducing conditions, intracellular survival of P. aeruginosa upon infection of HeLa cells, and twitching motility. The diverse phenotypes of the dsbA mutant are likely due to its defect in the folding of proteins that are involved in various biological processes, such as signal sensing, protein secretion, and defense against host clearing. In light of its effect on various virulence factors, DsbA could be an important target for the control of P. aeruginosa infections.

Growth phase-dependent invasion of Pseudomonas aeruginosa and its survival within HeLa cells.

ABSTRACT Clinical isolates of Pseudomonas aeruginosa are classified into invasive and noninvasive (cytolytic) strains. In a noninvasive PA103 background, ExoS and ExoT have recently been shown to function as anti-internalization factors. However, these two factors seemed not to have such a function in an invasive strain PAK background. In this study, using HeLa tissue culture cells, we observed that the internalization of invasive strain PAK is dependent on its growth phases, with the stationary-phase cells internalized about 100-fold more efficiently than the exponential-phase cells. This growth phase-dependent internalization was not observed in the noninvasive PA103 strain. Further analysis of various mutant derivatives of the invasive PAK and the noninvasive PA103 strains demonstrated that ExoS or ExoT that is injected into host cells by a type III secretion machinery functions as an anti-internalization factor in both types of strains. In correlation with the growth phase-dependent internalization, the invasive strain PAK translocates much higher amount of ExoS and ExoT into HeLa cells when it is in an exponential-growth phase than when it is in a stationary-growth phase, whereas the translocation of ExoT by the noninvasive strain PA103 is consistently high regardless of the growth phases, suggesting a difference in the regulatory mechanism of type III secretion between the two types of strains. Consistent with the invasive phenotype of the parent strain, an internalized PAK derivative survived well within the HeLa cells, whereas the viability of internalized PA103 derivative was dramatically decreased and completely cleared within 48 h. These results indicate that the invasive strains of P. aeruginosahave evolved the mechanism of intracellular survival, whereas the noninvasive P. aeruginosa strains have lost or not acquired the ability to survive within the epithelial cells.

Transfer of antibiotic resistance plasmids in pure and activated sludge cultures in the presence of environmentally representative micro-contaminant concentrations

The presence of antibiotics in the natural environment has been a growing issue. This presence could also account for the influence that affects microorganisms in such a way that they develop resistance against these antibiotics. The aim of this study was to evaluate whether the antibiotic resistant gene (ARG) plasmid transfer can be facilitated by the impact of 1) environmentally representative micro-contaminant concentrations in ppb (part per billion) levels and 2) donor-recipient microbial complexity (pure vs. mixed). For this purpose, the multidrug resistant plasmid, pB10, and Escherichia coli DH5α were used as a model plasmid and a model donor, respectively. Based on conjugation experiments with pure (Pseudomonas aeruginosa PAKexoT) and mixed (activated sludge) cultures as recipients, increased relative plasmid transfer frequencies were observed at ppb (μg/L) levels of tetracycline and sulfamethoxazole micro-contaminant exposure. When sludge, a more complex community, was used as a recipient, the increases of the plasmid transfer rate were always statistically significant but not always in P. aeruginosa. The low concentration (10 ppb) of tetracycline exposure led to the pB10 transfer to enteric bacteria, which are clinically important pathogens.

MKP1 Regulates the Induction of MUC5AC Mucin by Streptococcus pneumoniae Pneumolysin by Inhibiting the PAK4-JNK Signaling Pathway

Mucosal epithelial cells in the respiratory tract act as the first line of host innate defense against inhaled microbes by producing a range of molecules for clearance. In particular, epithelial mucins facilitate the mucociliary clearance by physically trapping the inhaled microbes. Up-regulation of mucin production thus represents an important host innate defense response against invading microbes. Excess mucin production, however, overwhelms the mucociliary clearance, resulting in defective mucosal defenses. Thus, tight regulation of mucin production is critical for maintaining an appropriate balance between beneficial and detrimental outcomes. Among various mechanisms, negative regulation plays an important role in tightly regulating mucin production. Here we show that the PAK4-JNK signaling pathway acted as a negative regulator for Streptococcus pneumoniae pneumolysin-induced MUC5AC mucin transcription. Moreover pneumolysin also selectively induced expression of MKP1 via a TLR4-dependent MyD88-TRAF6-ERK signaling pathway, which inhibited the PAK4-JNK signaling pathway, thereby leading to up-regulation of MUC5AC mucin production to maintain effective mucosal protection against S. pneumoniae infection. These studies provide novel insights into the molecular mechanisms underlying the tight regulation of mucin overproduction in the pathogenesis of airway infectious diseases and may lead to development of new therapeutic strategies.

Lens-free shadow image based high-throughput continuous cell monitoring technique.

A high-throughput continuous cell monitoring technique which does not require any labeling reagents or destruction of the specimen is demonstrated. More than 6000 human alveolar epithelial A549 cells are monitored for up to 72 h simultaneously and continuously with a single digital image within a cost and space effective lens-free shadow imaging platform. In an experiment performed within a custom built incubator integrated with the lens-free shadow imaging platform, the cell nucleus division process could be successfully characterized by calculating the signal-to-noise ratios (SNRs) and the shadow diameters (SDs) of the cell shadow patterns. The versatile nature of this platform also enabled a single cell viability test followed by live cell counting. This study firstly shows that the lens-free shadow imaging technique can provide a continuous cell monitoring without any staining/labeling reagent and destruction of the specimen. This high-throughput continuous cell monitoring technique based on lens-free shadow imaging may be widely utilized as a compact, low-cost, and high-throughput cell monitoring tool in the fields of drug and food screening or cell proliferation and viability testing.

Pseudomonas aeruginosa injects NDK into host cells through a type III secretion system

Pseudomonas aeruginosa 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, P. aeruginosa 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 P. aeruginosa 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.

Role of pneumococcal pneumolysin in the induction of an inflammatory response in human epithelial cells

Epithelial cells act as the first line of host defense against microorganisms by producing a range of molecules for clearance. Proinflammatory cytokines facilitate the clearance of invaders by the recruitment and activation of leukocytes. Upregulation of cytokine expression thus represents an important host innate defense response against invading microorganisms such as Streptococcus pneumoniae. Histological analysis of the airway revealed less leukocyte infiltration during the early stage of pneumococcal infection, when compared with nontypable Haemophilus influenzae (NTHi) infection. Here, we report that S. pneumoniae is less potent in inducing proinflammatory cytokine expression compared with NTHi. Among numerous virulence factors, pneumococcal pneumolysin was found to be the major factor responsible for the induction of inflammation. Interestingly, pneumolysin induces cytokine expression to a lesser extent at the early stage of infection, but becomes more potent in inducing inflammation at the late stage. Thus, this study reveals that pneumolysin induces the proinflammatory cytokine expression in a time-dependent manner.

Pseudomonas aeruginosa-induced IL-1β production is inhibited by Sophora flavescens via the NF-κB/inflammasome pathways.

The proinflammatory cytokine interleukin-1β plays an important role in protecting the host against airway infection; however, it can also trigger a massive influx of neutrophils into the airways, causing tissue damage. Anti-inflammatory treatments are particularly in demand for patients suffering from chronic inflammatory diseases. Sophora flavescens is a traditional herbal medicine used to reduce inflammation, but no study has examined its ability to block IL-1β production. Here, we show that S. flavescens reduced the Pseudomonas aeruginosa-induced expression of IL-1β by lung epithelial cells and macrophages. S. flavescens was also effective at reducing IL-1β production induced by either Staphylococcus aureus or phorbol 12-myristate 13-acetate, indicating that the effect is generalizable to diverse inflammatory stimuli. In addition, S. flavescens blocked the phosphorylation of IKKα/β, key upstream kinases involved in the degradation of IκBα, and the cleavage of caspase-1, a key component of the inflammasome. Thus, this study demonstrates that S. flavescens exerts its anti-inflammatory effects by blocking P. aeruginosa-mediated NF-κB/inflammasome activation and the subsequent production of IL-1β.

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

ABSTRACT 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 Pseudomonas aeruginosa 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 P. aeruginosa, through the combined effects of Ndk and flagellin, upregulates the expression of proinflammatory cytokines via the Akt/NF-κB signaling pathways.

The effects of antibiotics on the biofilm formation and antibiotic resistance gene transfer

AbstractRecently, a number of microcontaminants were found in aquatic environment and it raises the concerns about their effects to ecosystem. This study investigated if low levels of antibiotics can trigger the biofilm formation and result in the enhanced antibiotic gene transfer. For this purpose, the biofilm formation of enteric bacteria (Escherichia coli), environmental micro-organism (Pseudomonas aeruginosa), and their mixture by ppb level of antibiotics were investigated. In addition, the effects on the conjugation of E. coli with P. aeruginosa in the biofilm structure were also evaluated using biofilm colony-forming unit assay in 96-well plates and ELISA. Interestingly, at 100–1000 ppb, the mixed culture was able to reach its highest biofilm biomass and also form the highest number of transconjugants, which is greater than negative controls. This experiment shows that ppb levels of tetracycline and cephradine can alter the transfer rate of the pB10 plasmid among the biofilm biomass at rates 2–5 tim...