Shang-Chen Hsieh

A Mobile Quorum-Sensing System in Serratia marcescens

Quorum-sensing systems that have been widely identified in bacteria play important roles in the regulation of bacterial multicellular behavior by which bacteria sense population density to control various biological functions, including virulence. One characteristic of the luxIR quorum-sensing genes is their diverse and discontinuous distribution among proteobacteria. Here we report that the spnIR quorum-sensing system identified in the enterobacterium Serratia marcescens strain SS-1 is carried in a transposon, TnTIR, which has common characteristics of Tn3 family transposons and is mobile between chromosomes and plasmids of different enterobacterial hosts. SpnIR functions in the new host and was shown to negatively regulate the TnTIR transposition frequency. This finding may help reveal the horizontal transfer and evolutionary mechanism of quorum-sensing genes and alter the way that we perceive regulation of bacterial multicellular behavior.

Pirin Regulates Pyruvate Catabolism by Interacting with the Pyruvate Dehydrogenase E1 Subunit and Modulating Pyruvate Dehydrogenase Activity

The protein pirin, which is involved in a variety of biological processes, is conserved from prokaryotic microorganisms, fungi, and plants to mammals. It acts as a transcriptional cofactor or an apoptosis-related protein in mammals and is involved in seed germination and seedling development in plants. In prokaryotes, while pirin is stress induced in cyanobacteria and may act as a quercetinase in Escherichia coli, the functions of pirin orthologs remain mostly uncharacterized. We show that the Serratia marcescens pirin (pirin(Sm)) gene encodes an ortholog of pirin protein. Protein pull-down and bacterial two-hybrid assays followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization-tandem mass spectrometry analyses showed the pyruvate dehydrogenase (PDH) E1 subunit as a component interacting with the pirin(Sm) gene. Functional analyses showed that both PDH E1 subunit activity and PDH enzyme complex activity are inhibited by the pirin(Sm) gene in S. marcescens CH-1. The S. marcescens CH-1 pirin(Sm) gene was subsequently mutated by insertion-deletion homologous recombination. Accordingly, the PDH E1 and PDH enzyme complex activities and cellular ATP concentration increased up to 250%, 140%, and 220%, respectively, in the S. marcescens CH-1 pirin(Sm) mutant. Concomitantly, the cellular NADH/NAD(+) ratio increased in the pirin(Sm) mutant, indicating increased tricarboxylic acid (TCA) cycle activity. Our results show that the pirin(Sm) gene plays a regulatory role in the process of pyruvate catabolism to acetyl coenzyme A through interaction with the PDH E1 subunit and inhibiting PDH enzyme complex activity in S. marcescens CH-1, and they suggest that pirin(Sm) is an important protein involved in determining the direction of pyruvate metabolism towards either the TCA cycle or the fermentation pathways.

RssAB-FlhDC-ShlBA as a major pathogenesis pathway in Serratia marcescens.

ABSTRACT Serratia marcescens has long been recognized as an important opportunistic pathogen, but the underlying pathogenesis mechanism is not completely clear. Here, we report a key pathogenesis pathway in S. marcescens comprising the RssAB two-component system and its downstream elements, FlhDC and the dominant virulence factor hemolysin ShlBA. Expression of shlBA is under the positive control of FlhDC, which is repressed by RssAB signaling. At 37°C, functional RssAB inhibits swarming, represses hemolysin production, and promotes S. marcescens biofilm formation. In comparison, when rssBA is deleted, S. marcescens displays aberrant multicellularity favoring motile swarming with unbridled hemolysin production. Cellular and animal infection models further demonstrate that loss of rssBA transforms this opportunistic pathogen into hypervirulent phenotypes, leading to extensive inflammatory responses coupled with destructive and systemic infection. Hemolysin production is essential in this context. Collectively, a major virulence regulatory pathway is identified in S. marcescens.

Characterization of the dapA-nlpB Genetic Locus Involved in Regulation of Swarming Motility, Cell Envelope Architecture, Hemolysin Production, and Cell Attachment Ability in Serratia marcescens

ABSTRACT Swarming migration of Serratia marcescens requires both flagellar motility and cellular differentiation and is a population-density-dependent behavior. While the flhDC and quorum-sensing systems have been characterized as important factors regulating S. marcescens swarming, the underlying molecular mechanisms are currently far from being understood. Serratia swarming is thermoregulated and is characterized by continuous surface migration on rich swarming agar surfaces at 30°C but not at 37°C. To further elucidate the mechanisms, identification of specific and conserved regulators that govern the initiation of swarming is essential. We performed transposon mutagenesis to screen for S. marcescens strain CH-1 mutants that swarmed at 37°C. Analysis of a “precocious-swarming” mutant revealed that the defect in a conserved dapASm-nlpBSm genetic locus which is closely related to the synthesis of bacterial cell wall peptidoglycan is responsible for the aberrant swarming phenotype. Further complementation and gene knockout studies showed that nlpBSm, which encodes a membrane lipoprotein, NlpBSm, but not dapASm, is specifically involved in swarming regulation. On the other hand, dapASm but not nlpBSm is responsible for the determination of cell envelope architecture, regulation of hemolysin production, and cellular attachment capability. While the nlpBSm mutant showed similar cytotoxicity to its parent strain, the dapASm mutant significantly increased in cytotoxicity. We present evidence that DapASm is involved in the determination of cell-envelope-associated phenotypes and that NlpBSm is involved in the regulation of swarming motility.

Resveratrol ameliorates Serratia marcescens-induced acute pneumonia in rats

Serratia marcescens is an important nosocomial pathogen, which has been especially problematic as a cause of hospital‐acquired pneumonia in the past two decades. Treatment of S. marcescens‐related infections has been limited by emergence of multiple drug‐resistant strains. Thus, the development of alternative agents for the prevention and treatment of Serratia infection is urgently needed. Resveratrol (RSV) is a compound with diverse biological effects including anti‐cancer, anti‐inflammation, anti‐diabetes, and cancer chemoprevention. Whether RSV has in vivo prophylactic or therapeutic potential against infection remains uncharacterized. In the present study, we used a murine acute pneumonia model initiated by intratracheal application of S. marcescens to evaluate whether RSV possesses anti‐infection properties. We showed that pretreatment with RSV for 3 days markedly increased alveolar macrophage infiltration, elevated NK cell activity, and decreased bacterial burden in the infected lung with a subsequent decrease in mortality. These effects were associated with significantly less‐severe inflammatory phenotypes in lung tissue and bronchoalveolar lavage fluid, including reduced neutrophil infiltration of the lungs, reduced phagocytosis activity, and reduced secretion of cytokines such as TNF‐α, IL‐1β, and IL‐6. To further characterize the underlying mechanism responsible for these effects of RSV, LPS derived from S. marcescens was used to induce acute pneumonia in rats, with or without RSV pretreatment. RSV was shown to ameliorate acute pneumonia via inhibition of the NF‐κB signaling pathway, including inhibition of IκBα phosphorylation and subsequent NF‐κB activation. These findings suggest that RSV might be beneficial as a prophylactic treatment in patients at risk of an episode of S. marcescens‐induced acute pneumonia.

Rapid identification of Mycobacterium tuberculosis infection by a new array format-based surface plasmon resonance method

Tubercle bacillus [TB] is one of the most important chronic infectious diseases that cause millions of deaths annually. While conventional smear microscopy and culture methods are widely used for diagnosis of TB, the former is insensitive, and the latter takes up to 6 to 8 weeks to provide a result, limiting the value of these methods in aiding diagnosis and intermediate decisions on treatment. Therefore, a rapid detection method is essential for the diagnosis, prognosis assessment, and recurrence monitoring. A new surface plasmon resonance [SPR] biosensor based on an array format, which allowed immobilizing nine TB antigens onto the sensor chip, was constructed. Simultaneous determination of multiple TB antibodies in serum had been accomplished with this array-based SPR system. The results were compared with enzyme-linked immunosorbent assay, a conventional immunological method. Array-based SPR showed more advantages in providing label-free and real-time detection. Additionally, the high sensitivity and specificity for the detection of TB infection showed its potential for future development of biosensor arrays for TB diagnosis.

Biochemical Characterization of RssA-RssB, a Two-Component Signal Transduction System Regulating Swarming Behavior in Serratia marcescens

Our previous study had identified a pair of potential two-component signal transduction proteins, RssA-RssB, involved in the regulation of Serratia marcescens swarming. When mutated, both rssA and rssB mutants showed precocious swarming phenotypes on LB swarming agar, whereby swarming not only occurred at 37 degrees C but also initiated on a surface of higher agar concentration and more rapidly than did the parent strain at 30 degrees C. In this study, we further show that the predicted sensor kinase RssA and the response regulator RssB bear characteristics of components of the phosphorelay signaling system. In vitro phosphorylation and site-directed mutagenesis assays showed that phosphorylated RssA transfers the phosphate group to RssB and that histidine 248 and aspartate 51 are essential amino acid residues involved in the phosphotransfer reactions in RssA and RssB, respectively. Accordingly, while wild-type rssA could, the mutated rssA(H248A) in trans could not complement the precocious swarming phenotype of the rssA mutant. Although RssA-RssB regulates expressions of shlA and ygfF of S. marcescens (ygfF(Sm)), in vitro DNA-binding assays showed that the phosphorylated RssB did not bind directly to the promoter regions of these two genes but bound to its own rssB promoter. Subsequent assays located the RssB binding site within a 63-bp rssB promoter DNA region and confirmed a direct negative autoregulation of the RssA-RssB signaling pathway. These results suggest that when activated, RssA-RssB acts as a negative regulator for controlling the initiation of S. marcescens swarming.

Rapid identification of Mycobacterium avium clinical isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

BACKGROUND Rapid and accurate discrimination of Mycobacterium avium from other mycobacteria is essential for appropriate therapeutic management and timely intervention for infection control. However, routine clinical identification methods for M. avium are both time consuming and labor intensive. In the present study, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to identify specific cellular protein pattern for rapid identification of M. avium isolates. METHODS A total of 40 clinically relevant Mycobacterium strains comprising 13 distinct species were enrolled for the MALDI-TOF MS identification. A 10-minute extraction-free examination procedure was set up to obtain mass spectral fingerprints from whole bacterial cells. RESULTS The characteristic mass spectral peak patterns in the m/z (mass/charge ratio) range of 5-20 kDa can be obtained within 10 minutes. The species-specific mass spectra for M. avium is identified and can be differentiated from as Mycobacterium strains. This technique shortens and simplifies the identification procedure of MALDI-TOF MS and may further extend the mycobacterial MALDI-TOF MS database. CONCLUSION Simplicity and rapidity of identification procedures make MALDI-TOF MS an attractive platform in routine identification of mycobacteria. MALDI-TOF MS is applicable for rapid discrimination of M. avium from other Mycobacterium species, and shows its potential for clinical application.