Keehoon Lee

Macrolide Treatment for Mycobacterium abscessus and Mycobacterium massiliense Infection and Inducible Resistance

RATIONALE Macrolides, such as clarithromycin (CLR) and azithromycin (AZM), are frequently the only oral antibiotics that are active against Mycobacterium abscessus and M. massiliense infections. OBJECTIVES To compare the activity of CLR and AZM in experimental models. METHODS We compared the treatment efficacies of CLR and AZM and determined the correlation between efficacy and induced erythromycin ribosome methyltransferase gene (erm)(41) expression in experimental models of M. abscessus and M. massiliense infections. MEASUREMENTS AND MAIN RESULTS In all tested M. abscessus isolates, a high level of inducible CLR resistance developed (minimal inhibitory concentration [MIC] on Day 3 versus Day 14; P < 0.001). Whereas the AZM MIC increased on Day 14 (P < 0.01 versus Day 3), the level was significantly lower than the CLR MIC on Day 14 (P < 0.001). However, the MICs of CLR and AZM for the M. massiliense isolates did not change. Compared with CLR, AZM presented greater antibiotic activity against M. abscessus in vitro, ex vivo, and in vivo (P < 0.05), whereas both macrolides were comparably effective against M. massiliense. In M. abscessus infection, the level of erm(41) expression was higher after exposure to CLR than after exposure to AZM (P < 0.001). Experiments using an erm(41)-knockout M. abscessus mutant and an M. massiliense transformant expressing M. abscessus erm(41) confirmed that erm(41) was responsible for inducible CLR resistance. CONCLUSIONS CLR induces greater erm(41) expression and thus higher macrolide resistance than AZM in M. abscessus infection. AZM may be more effective against M. abscessus, whereas both macrolides appear to be equally effective against M. massiliense.

Mycobacterium tuberculosis RpfB drives Th1-type T cell immunity via a TLR4-dependent activation of dendritic cells

The failure of Mycobacterium bovis BCG as a TB vaccine against TB reactivation suggests that latency‐associated proteins should be included in alternative TB vaccine development. Further, antigens known to generate protective immunity against the strong Th1 stimulatory response to reactivated TB should be included in novel vaccine design. Recent studies have emphasized the importance of Rpfs from Mycobacterium tuberculosis in the reactivation process and cellular immunity. However, little is known about how RpfB mediates protective immunity against M. tuberculosis. Here, we investigated the functional roles and signaling mechanisms of RpfB in DCs and its implications in the development of T cell immunity. DCs treated with RpfB displayed features of mature and functional status, with elevated expression of cell surface molecules (CD80, CD86, and MHC class I and II) and proinflammatory cytokine production (TNF‐α, IL‐1β, IL‐6, and IL‐12p70). Activation of DCs was mediated by direct binding of RpfB to TLR4, followed by MyD88/TRIF‐dependent signaling to MAPKs and NF‐κB signaling pathways. Specifically, we found that the RpfB G5 domain is the most important part in RpfB binding to TLR4. RpfB‐treated DCs effectively polarized naïve CD4+ and CD8+ T cells to secrete IFN‐γ and IL‐2. Importantly, RpfB induced the expansion of memory CD4+/CD8+CD44highCD62Llow T cells in the spleen of M. tuberculosis‐infected mice. Our data suggest that RpfB regulates innate immunity and activates adaptive immunity through TLR4, a finding that may help in the design of more effective vaccines.

Mycobacterium paratuberculosis CobT Activates Dendritic Cells via Engagement of Toll-like Receptor 4 Resulting in Th1 Cell Expansion

Background: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne and Crohn diseases. Results: CobT contributes to the development of T cell immunity through the activation of dendritic cells (DC). Conclusion: CobT is a novel DC maturation-inducing antigen that drives Th1 polarized-naive/memory T cell expansion. Significance: CobT can be an important candidate for the development of vaccine and a IFN-γ-based diagnostic tool. Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne disease in animals and MAP involvement in human Crohn disease has been recently emphasized. Evidence from M. tuberculosis studies suggests mycobacterial proteins activate dendritic cells (DCs) via Toll-like receptor (TLR) 4, eventually determining the fate of immune responses. Here, we investigated whether MAP CobT contributes to the development of T cell immunity through the activation of DCs. MAP CobT recognizes TLR4, and induces DC maturation and activation via the MyD88 and TRIF signaling cascades, which are followed by MAP kinases and NF-κB. We further found that MAP CobT-treated DCs activated naive T cells, effectively polarized CD4+ and CD8+ T cells to secrete IFN-γ and IL-2, but not IL-4 and IL-10, and induced T cell proliferation. These data indicate that MAP CobT contributes to T helper (Th) 1 polarization of the immune response. MAP CobT-treated DCs specifically induced the expansion of CD4+/CD8+CD44highCD62Llow memory T cells in the mesenteric lymph node of MAP-infected mice in a TLR4-dependent manner. Our results indicate that MAP CobT is a novel DC maturation-inducing antigen that drives Th1 polarized-naive/memory T cell expansion in a TLR4-dependent cascade, suggesting that MAP CobT potentially links innate and adaptive immunity against MAP.

Protective role of gut commensal microbes against intestinal infections

The human gastrointestinal tract is colonized by multitudes of microorganisms that exert beneficial effects on human health. Mounting evidence suggests that intestinal microbiota contributes to host resistance against enteropathogenic bacterial infection. However, molecular details that account for such an important role has just begun to be understood. The commensal microbes in the intestine regulate gut homeostasis through activating the development of host innate immunity and producing molecules with antimicrobial activities that directly inhibit propagation of pathogenic bacteria. Understanding the protective roles of gut microbiota will provide a better insight into the molecular basis that underlies complicated interaction among host-pathogen-symbiont. In this review, we highlighted recent findings that help us broaden our knowledge of the intestinal ecosystem and thereby come up with a better strategy for combating enteropathogenic infection.

A single gene of a commensal microbe affects host susceptibility to enteric infection

Indigenous microbes inside the host intestine maintain a complex self-regulating community. The mechanisms by which gut microbes interact with intestinal pathogens remain largely unknown. Here we identify a commensal Escherichia coli strain whose expansion predisposes mice to infection by Vibrio cholerae, a human pathogen. We refer to this strain as ‘atypical’ E. coli (atEc) because of its inability to ferment lactose. The atEc strain is resistant to reactive oxygen species (ROS) and proliferates extensively in antibiotic-treated adult mice. V. cholerae infection is more severe in neonatal mice transplanted with atEc compared with those transplanted with a typical E. coli strain. Intestinal ROS levels are decreased in atEc-transplanted mice, favouring proliferation of ROS-sensitive V. cholerae. An atEc mutant defective in ROS degradation fails to facilitate V. cholerae infection when transplanted, suggesting that host infection susceptibility can be regulated by a single gene product of one particular commensal species.

Differential immune responses to Segniliparus rotundus and Segniliparus rugosus infection and analysis of their comparative virulence profiles.

Two closely related bacterial species, Segniliparus rotundus and Segniliparus rugosus, have emerged as important human pathogens, but little is known about the immune responses they elicit or their comparative pathophysiologies. To determine the virulence and immune responses of the two species, we compared their abilities to grow in phagocytic and non-phagocytic cells. Both species maintained non-replicating states within A549 epithelial cells. S. rugosus persisted longer and multiplied more rapidly inside murine bone marrow-derived macrophages (BMDMs), induced more pro-inflammatory cytokines, and induced higher levels of macrophage necrosis. Activation of BMDMs by both species was mediated by toll-like receptor 2 (TLR2), followed by mitogen-activated protein kinases (MAPK) and nuclear factor κB (NF-κB) signaling pathways, indicating a critical role for TLR2 in Segniliparus-induced macrophage activation. S. rugosus triggered faster and stronger activation of MAPK signaling and IκB degradation, indicating that S. rugosus induces more pro-inflammatory cytokines than S. rotundus. Multifocal granulomatous inflammations in the liver and lung were observed in mice infected with S. rugosus, but S. rotundus was rapidly cleared from all organs tested within 15 days post-infection. Furthermore, S. rugosus induced faster infiltration of innate immune cells such as neutrophils and macrophages to the lung than S. rotundus. Our results suggest that S. rugosus is more virulent and induces a stronger immune response than S. rotundus.

Molecular determinants of the matrix thickening of a dual-species Pseudomonas aeruginosa and Enterococcus faecalis biofilm

ABSTRACT Biofilms are microbial communities that inhabit various surfaces and are surrounded by extracellular matrices (ECMs). Clinical microbiologists have shown that the majority of chronic infections are caused by biofilms, following the introduction of the first biofilm infection model by J. W. Costerton and colleagues (J. Lam, R. Chan, K. Lam, and J. W. Costerton, Infect Immun 28:546–556, 1980). However, treatments for chronic biofilm infections are still limited to surgical removal of the infected sites. Pseudomonas aeruginosa and Enterococcus faecalis are two frequently identified bacterial species in biofilm infections; nevertheless, the interactions between these two species, especially during biofilm growth, are not clearly understood. In this study, we observed phenotypic changes in a dual-species biofilm of P. aeruginosa and E. faecalis, including a dramatic increase in biofilm matrix thickness. For clear elucidation of the spatial distribution of the dual-species biofilm, P. aeruginosa and E. faecalis were labeled with red and green fluorescence, respectively. E. faecalis was located at the lower part of the dual-species biofilm, while P. aeruginosa developed a structured biofilm on the upper part. Mutants with altered exopolysaccharide (EPS) productions were constructed in order to determine the molecular basis for the synergistic effect of the dual-species biofilm. Increased biofilm matrix thickness was associated with EPSs, not extracellular DNA. In particular, Pel and Psl contributed to interspecies and intraspecies interactions, respectively, in the dual-species P. aeruginosa and E. faecalis biofilm. Accordingly, targeting Pel and Psl might be an effective part of eradicating P. aeruginosa polymicrobial biofilms. IMPORTANCE Chronic infection is a serious problem in the medical field. Scientists have observed that chronic infections are closely associated with biofilms, and the vast majority of infection-causing biofilms are polymicrobial. Many studies have reported that microbes in polymicrobial biofilms interact with each other and that the bacterial interactions result in elevated virulence, in terms of factors, such as infectivity and antibiotic resistance. Pseudomonas aeruginosa and Enterococcus faecalis are frequently isolated pathogens in chronic biofilm infections. Nevertheless, while both bacteria are known to be agents of numerous nosocomial infections and can cause serious diseases, interactions between the bacteria in biofilms have rarely been examined. In this investigation, we aimed to characterize P. aeruginosa and E. faecalis dual-species biofilms and to determine the molecular factors that cause synergistic effects, especially on the matrix thickening of the biofilm. We suspect that our findings will contribute to the development of more efficient methods for eradicating polymicrobial biofilm infections.

Minimizing MR Gradient and RF pulse Artefacts on ECG Signals for MRI Gating based on an Adaptive real-time digital filter

In Magnetic resonance imaging(MRI), imaging a moving organ such as the heart requires a trigger so that successive scans can be synchronized. In the case of cardiac imaging, the QRS complex of ECG is used as a trigger signal for MRI scan. But, gradient and RF(radio frequency) artifacts which are caused to static and dynamic field in MRI scanner cause interference in the ECG. Also, the signal shape of theses artifacts can be similar to the QRS-complex, causing possible misinterpretation during patient monitoring and false gating of the MRI. In case of using general FIR or IIR band-pass filters for minimizing the artifacts, artifact-reduction-ratio is not excellent. So, an adaptive real-time digital filter is proposed for reduction of noise by gradient and RF(radio frequency) artifacts. The proposed filter for MRI-Gating is based on the noise-canceller with NLMS(Normalized Least Mean Square) algorithm. The reference signals of the adaptive noise canceller are a combination of the noisy three channel ECG signals. Various tests were done on normal volunteers in various scenarios[SE(spin echo), FSE(fast spin echo), TR(transition time), TX-Gain, RX-gain, Spin-echo-degree, soon]. The noise canceller’s performance was measured offline, simulating real-time processing by point-by-point operations. In conclusions, the proposed method showed the acceptable quality of ECG signal with sufficient SNR for gating the MRI and possibility of real time implementation

Correlative-encoded (CE) FSK for use in RF home networking

A new power and bandwidth efficient modem technique, correlative encoded FSK (CEFSK), is proposed for use in RF home networking system. Power spectral densities (PSD) and BER performance of CEFSK are analyzed and compared with those of GFSK via computer simulation. A new fractional multi-bit differential detection (FMDD) technique is also proposed in order to improve the BER performance of CEFSK. Our simulation results show that CEFSK has a spectral efficiency comparable to GFSK, and that BER performance of CEFSK using 5-branch FMDD technique outperforms that of GFSK by 2.0 dB at a BER=1 /spl times/ 10/sup -4/ in AWGN and multipath fading channels.

A Genetic Screen Reveals Novel Targets to Render Pseudomonas aeruginosa Sensitive to Lysozyme and Cell Wall-Targeting Antibiotics

Pseudomonas aeruginosa is capable of establishing airway infections. Human airway mucus contains a large amount of lysozyme, which hydrolyzes bacterial cell walls. P. aeruginosa, however, is known to be resistant to lysozyme. Here, we performed a genetic screen using a mutant library of PAO1, a prototype P. aeruginosa strain, and identified two mutants (ΔbamB and ΔfabY) that exhibited decrease in survival after lysozyme treatment. The bamB and fabY genes encode an outer membrane assembly protein and a fatty acid synthesis enzyme, respectively. These two mutants displayed retarded growth in the airway mucus secretion (AMS). In addition, these mutants exhibited reduced virulence and compromised survival fitness in two different in vivo infection models. The mutants also showed susceptibility to several antibiotics. Especially, ΔbamB mutant was very sensitive to vancomycin, ampicillin, and ceftazidime that target cell wall synthesis. The ΔfabY displayed compromised membrane integrity. In conclusion, this study uncovered a common aspect of two different P. aeruginosa mutants with pleiotropic phenotypes, and suggests that BamB and FabY could be novel potential drug targets for the treatment of P. aeruginosa infection.