Hwei-Ling Peng

Liver-cell patterning Lab Chip: mimicking the morphology of liver lobule tissue

A lobule-mimetic cell-patterning technique for on-chip reconstruction of centimetre-scale liver tissue of heterogeneous hepatic and endothelial cells via an enhanced field-induced dielectrophoresis (DEP) trap is demonstrated and reported. By mimicking the basic morphology of liver tissue, the classic hepatic lobule, the lobule-mimetic-stellate-electrodes array was designed for cell patterning. Through DEP manipulation, well-defined and enhanced spatial electric field gradients were created for in-parallel manipulation of massive individual cells. With this liver-cell patterning labchip design, the original randomly distributed hepatic and endothelial cells inside the microfluidic chamber can be manipulated separately and aligned into the desired pattern that mimicks the morphology of liver lobule tissue. Experimental results showed that both hepatic and endothelial cells were orderly guided, snared, and aligned along the field-induced orientation to form the lobule-mimetic pattern. About 95% cell viability of hepatic and endothelial cells was also observed after cell-patterning demonstration via a fluorescent assay technique. The liver function of CYP450-1A1 enzyme activity showed an 80% enhancement for our engineered liver tissue (HepG2+HUVECs) compared to the non-patterned pure HepG2 for two-day culturing.

RmpA2, an Activator of Capsule Biosynthesis in Klebsiella pneumoniae CG43, Regulates K2 cps Gene Expression at the Transcriptional Level

The rmpA2 gene, which encodes an activator for capsular polysaccharide (CPS) synthesis, was isolated from a 200-kb virulence plasmid of Klebsiella pneumoniae CG43. Based on the sequence homology with LuxR at the carboxyl-terminal DNA-binding motif, we hypothesized that RmpA2 exerts its effect by activating the expression of cps genes that are responsible for CPS biosynthesis. Two luxAB transcriptional fusions, each containing a putative promoter region of the K. pneumoniae K2 cps genes, were constructed and were found to be activated in the presence of multicopy rmpA2. The activation is likely due to direct binding of RmpA2 to the cps gene promoter through its C-terminal DNA binding motif. Moreover, the loss of colony mucoidy in a K. pneumoniae strain deficient in RcsB, a regulator for cps gene expression, could be recovered by complementing the strain with a multicopy plasmid carrying rmpA2. The CPS production in Lon protease-deficient K. pneumoniae significantly increased, and the effect was accompanied by an increase of RmpA2 stability. The expression of the rmpA2 gene was negatively autoregulated and could be activated when the organism was grown in M9 minimal medium. An IS3 element located upstream of the rmpA2 was required for the full activation of the rmpA2 promoter. In summary, our results suggest that the enhancement of K2 CPS synthesis in K. pneumoniae CG43 by RmpA2 can be attributed to its transcriptional activation of K2 cps genes, and the expression level of rmpA2 is autoregulated and under the control of Lon protease.

Novel microchip for in situ TEM imaging of living organisms and bio-reactions in aqueous conditions

A novel and disposable microchip (K-kit) with SiO(2) nano-membranes was developed and used as a specimen kit for in situ imaging of living organisms in an aqueous condition using transmission electron microscopy (TEM) without equipment modification. This K-kit enabled the successful TEM observation of living Escherichia coli cells and the tellurite reduction process in Klebsiella pneumoniae. The K. pneumoniae and Saccharomyces cerevisiae can stay alive in K-kit after continuous TEM imaging for up to 14 s and 42 s, respectively. Besides, different tellurite reduction profiles in cells grown in aerobic and anaerobic environments can be clearly revealed. These results demonstrate that the K-kit developed in this paper can be useful for observing living organisms and monitoring biological processes in situ.

Identification of Genes Induced In Vivo during Klebsiella pneumoniae CG43 Infection

ABSTRACT A novel in vivo expression technology (IVET) was performed to identify Klebsiella pneumoniae CG43 genes that are specifically expressed during infection of BALB/c mice. The IVET employed a UDP glucose pyrophosphorylase (galU)-deficient mutant of K. pneumoniae which is incapable of utilizing galactose and synthesizing capsular polysaccharide, as demonstrated by its low virulence to BALB/c mice and a white nonmucoid colony morphology on MacConkey-galactose agar. By using a functionalgalU gene as the reporter, an IVE promoter could render thegalU mutant virulent while maintaining the white nonmucoid colony phenotype. A total of 20 distinct sequences were obtained through the in vivo selection. Five of them have been identified previously as virulence-associated genes in other pathogens, while another five with characterized functions are involved in regulation and transportation of nutrient uptake, biosynthesis of isoprenoids, and protein folding. No known functions have been attributed to the other 10 sequences. We have also demonstrated that 2 of the 20 IVE genes turn on under iron deprivation, whereas the expression of another five genes was found to be activated in the presence of paraquat, a superoxide generator.

Characterization of the Histidine-containing Phosphotransfer Protein B-mediated Multistep Phosphorelay System in Pseudomonas aeruginosa PAO1

Certain bacterial two-component sensor kinases possess a histidine-containing phosphotransfer (Hpt) domain to carry out a multistep phosphotransferring reaction to a cognate response regulator. Pseudomonas aeruginosa PAO1 contains three genes that encode proteins with an Hpt domain but lack a kinase domain. To identify the sensor kinase coupled to these Hpt proteins, a phosphorelay profiling assay was performed. Among the 12 recombinant orphan sensor kinases tested, 4 of these sensors (PA1611, PA1976, PA2824, and RetS) transferred the phosphoryl group to HptB (PA3345). The in vivo interaction between HptB and each of the sensors was also confirmed using the bacterial two-hybrid assay. Interestingly, the phosphoryl groups from these sensors all appeared to be transferred via HptB to PA3346, a novel phosphatase consisting of an N-terminal receiver domain and a eukaryotic type Ser/Thr phosphatase domain, and resulted in a significant increase of its phosphatase activity. The subsequent reverse transcription-PCR analysis revealed an operon structure of hptB-PA3346–PA3347, suggesting a coordinate expression of the three genes to carry out a signal transduction. The possibility was supported by the analysis showing PA3347 is able to be phosphorylated on Ser-56, and this phosphoryl group could be removed by PA3346 protein. Finally, analysis of PA3346 and PA3347 gene knock-out mutants revealed that these genes are associated with bacterial swarming activity and biofilm formation. Together, these results disclose a novel multistep phosphorelay system that is essential for P. aeruginosa to respond to a wide spectrum of environmental signals.

Genetic Requirements for Klebsiella pneumoniae-Induced Liver Abscess in an Oral Infection Model

ABSTRACT Klebsiella pneumoniae is the predominant pathogen of primary liver abscess. However, our knowledge regarding the molecular basis of how K. pneumoniae causes primary infection in the liver is limited. We established an oral infection model that recapitulated the characteristics of liver abscess and conducted a genetic screen to identify the K. pneumoniae genes required for the development of liver abscess in mice. Twenty-eight mutants with attenuated growth in liver or spleen samples out of 2,880 signature-tagged mutants that produced the wild-type capsule were identified, and genetic loci which were disrupted in these mutants were identified to encode products with roles in cellular metabolism, adhesion, transportation, gene regulation, and unknown functions. We further evaluated the virulence attenuation of these mutants in independent infection experiments and categorized them accordingly into three classes. In particular, the class I and II mutant strains exhibited significantly reduced virulence in mice, and most of these strains were not detected in extraintestinal tissues at 48 h after oral inoculation. Interestingly, the mutated loci of about one-third of the class I and II mutant strains encode proteins with regulatory functions, and the transcript abundances of many other genes identified in the same screen were markedly changed in these regulatory mutant strains, suggesting a requirement for genetic regulatory networks for translocation of K. pneumoniae across the intestinal barrier. Furthermore, our finding that preimmunization with certain class I mutant strains protected mice against challenge with the wild-type strain implied a potential application for these strains in prophylaxis against K. pneumoniae infections.

Identification of Genes Present Specifically in a Virulent Strain of Klebsiella pneumoniae

ABSTRACT Klebsiella pneumoniae is a common cause of septicemia and urinary tract infections. The PCR-supported genomic subtractive hybridization was employed to identify genes specifically present in a virulent strain of K. pneumoniae. Analysis of 25 subtracted DNA clones has revealed 19 distinct nucleotide sequences. Two of the sequences were found to be the genes encoding the transposase of Tn3926 and a capsule polysaccharide exporting enzyme. Three sequences displayed moderate homology with bvgAS, which encodes a two-component signal transduction system in Bordetella pertussis. The rest of the sequences did not exhibit homology with any known genes. The distribution of these novel sequences varied greatly in K. pneumoniae clinical isolates, reflecting the heterogeneous nature of the K. pneumoniae population.

Surface rigidity change of Escherichia coli after filamentous bacteriophage infection.

In this study, the feasibility using atomic force microscopy (AFM) to study the interaction between bacteriophages (phages) and bacteria in situ was demonstrated here. Filamentous phage M13 specifically infects the male Escherichia coli, which expresses F-pili. After infection, E. coli become fragile and grows at a slower rate. AFM provides a powerful tool for investigating these changes in a near-physiological environment. Using high-resolution AFM in phosphate-buffered saline, the damage to the lipopolysaccharide (LPS) layer on the outer membrane of the M13 phage-infected E. coli was observed. The membrane became smoother and more featureless compared to those that were not infected. Besides, the force-distance (f-d) curves were measured to reveal the surface rigidity change in E. coli after M13 phage infection. The effective spring constant and Youngs modulus of E. coli decreased after M13 phage infection. Furthermore, the AFM tip was pressed against E. coli to study the response of E. coli under load before and after M13 phage infection. The results showed that after infection E. coli became less rigid and the membrane was also damaged. However, the stiffness changes, including the spring constant and Youngs modulus of E. coli, are negligible after M13 phage infection compared with those in previous reports, which may be one of the reasons that E. coli still can maintain its viability after filamentous phage infection.

Comparative Analysis of Two UDP-glucose Dehydrogenases in Pseudomonas aeruginosa PAO1

UDP-glucose dehydrogenase (UGDH) catalyzes a two-step NAD+-dependent oxidation of UDP-glucose to produce UDP-glucuronic acid, which is a common substrate for the biosynthesis of exopolysaccharide. Searching the Pseudomonas aeruginosa PAO1 genome data base for a UGDH has helped identify two open reading frames, PA2022 and PA3559, which may encode a UGDH. To elucidate their enzymatic identity, the two genes were cloned and overexpressed in Escherichia coli, and the recombinant proteins were purified. Both the gene products are active as dimers and are capable of utilizing UDP-glucose as a substrate to generate UDP-glucuronic acid. The Km values of PA2022 and PA3559 for UDP-glucose are ∼0.1 and 0.4 mm, whereas the Km values for NAD+ are 0.5 and 2.0 mm, respectively. Compared with PA3559, PA2022 exhibits broader substrate specificity, utilizing TDP-glucose and UDP-N-acetylglucosamine with one-third the velocity of that with UDP-glucose. The PA2022 mutant and PA2022-PA3559 double mutant, but not the PA3559 mutant, are more susceptible to chloramphenicol, cefotaxime, and ampicillin. The PA3559 mutant, however, shows a reduced resistance to polymyxin B compared with wild type PAO1. Finally, real time PCR analysis indicates that PA3559 is expressed primarily in low concentrations of Mg2+, which contrasts with the constitutive expression of PA2022. Although both the enzymes catalyze the same reaction, their enzymatic properties and gene expression profiles indicate that they play distinct physiological roles in P. aeruginosa, as reflected by different phenotypes displayed by the mutants.

Regulation of the Klebsiella pneumoniae Kpc fimbriae by the site-specific recombinase KpcI

In the genome of Klebsiella pneumoniae NTUH-K2044, nine fimbrial gene clusters were identified. Besides type 1 and type 3 fimbriae, the others are novel and were named Kpa, Kpb, Kpc, Kpd, Kpe, Kpf and Kpg fimbriae. Prevalence analysis among 105 K. pneumoniae clinical isolates revealed that the kpc genes were highly associated with the K1 serotype isolates. Induced expression of the recombinant kpcABCD genes in Escherichia coli resulted in Kpc fimbriation and increased biofilm formation. A putative site-specific recombinase encoding gene kpcI and a 302 bp intergenic DNA flanked by 11 bp inverted repeats, namely kpcS, were identified in the upstream region of the kpcABCD genes. Using LacZ as the reporter, a dramatic difference in promoter activity of kpcS in two different orientations was observed and accordingly assigned as ON and OFF phase. kpcI expression was found to be able to invert kpcS in trans from phase ON to OFF and vice versa. Using the two-plasmid system, expression of kpcA, encoding the major component of the Kpc fimbriae, could be observed upon the induced expression of kpcI. These results indicate that KpcI is involved in the regulation of Kpc fimbriation in a phase-variable manner.