Yi-Chyi Lai

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.

RmpA Regulation of Capsular Polysaccharide Biosynthesis in Klebsiella pneumoniae CG43

Sequence analysis of the large virulence plasmid pLVPK in Klebsiella pneumoniae CG43 revealed the presence of another mucoid factor encoding gene rmpA besides rmpA2. Promoter activity measurement indicated that the deletion of rmpA reduced K2 capsular polysaccharide (CPS) biosynthesis, resulting in decreased colony mucoidy and virulence in mice. Introduction of a multicopy plasmid carrying rmpA restored CPS production in the rmpA or rmpA2 mutant but not in the rcsB mutant. Transformation of the rmpA deletion mutant with an rcsB-carrying plasmid also failed to enhance CPS production, suggesting that a cooperation of RmpA with RcsB is required for regulatory activity. This was further corroborated by the demonstration of in vivo interaction between RmpA and RcsB using two-hybrid analysis and coimmunoprecipitation analysis. A putative Fur binding box was only found at the 5 noncoding region of rmpA. The promoter activity analysis indicated that the deletion of fur increased the rmpA promoter activity. Using electrophoretic mobility shift assay, we further demonstrated that Fur exerts its regulatory activity by binding directly to the promoter. As a result, the fur deletion mutant exhibited an increase in colony mucoidy, CPS production, and virulence in mice. In summary, our results suggested that RmpA activates CPS biosynthesis in K. pneumoniae CG43 via an RcsB-dependent manner. The expression of rmpA is regulated by the availability of iron and is negatively controlled by Fur.

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.

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.

Function and sequence analyses of tumor suppressor gene p53 of CHO.K1 cells.

The tumor suppressor gene p53 plays an important role in guarding genomic integrity. When induced in response to environmental results, the gene product of p53 functions as a transcription factor to transactivate genes involved in arresting the cell cycle and as a facilitator of DNA repair. In contrast, the status of p53 in Chinese hamster ovary (CHO) cells, commonly used as a model system for various studies including those involving the cell cycle and transformation, remains an enigma. In this study, the function and sequence of p53 in CHO.K1 cells were investigated. The level of p53 proteins was elevated on ultraviolet (UV) irradiation of the cells, and the proteins formed specific complexes as probed with DNA containing p53-binding sequences. Its activities toward responsive promoters were inducible by UV in a dose-dependent manner. Although p53 in CHO.K1 contained a single missense mutation at codon 211, the mutation apparently had no effect on the functional properties of the protein. The CHO.K1 cells on X-ray irradiation failed to arrest at G1 phase even when the cells were transfected with a wildtype human p53 gene, indicating that the failure probably was not caused by dysfunction of its p53, but by some other mechanism. This result is consistent with the finding that p21(Waf1/Cip1) is undetectable in UV-treated CHO.K1 cells, whereas Gadd45 is induced by UV light in the cells.

UV inducibility of rat proliferating cell nuclear antigen gene promoter

Proliferating cell nuclear antigen (PCNA), also known as a cofactor of DNA polymerase δ, is required for eukaryotic cell DNA synthesis and nucleotide excision repair. Expression of PCNA gene is growth‐regulated and UV inducible. In our previous study, we have observed that the rat PCNA promoter has the serum responsiveness. In this study, we demonstrate its UV inducibility in CHO.K1 cells. The UV induction of the rat PCNA promoter activity was dose‐dependent in the cells synchronized at different phases. In addition, the sequences of the promoter responsible for the UV inducibility were delimited to the region between nucleotides −70 and +125, which contains an AP‐1 site and a downstream proximal ATF/CRE site. While mutation of the AP‐1 site abrogated the UV inducibility, mutation of the ATF/CRE site enhanced the UV inducibility, suggesting that the two sites play different roles in the UV induction of the promoter. In addition, the role of p53 in the UV induction of rat PCNA promoter was investigated. We found that exogenous p53 was unable to mimic the UV irradiation to induce rat PCNA promoter and that the UV induction of the rat PCNA promoter was seen in p53 deficient cells. Therefore, it is unlikely that the UV induction of the rat PCNA promoter is p53 dependent. J. Cell. Biochem. 73:423–432, 1999.

Serum responsiveness of the rat PCNA promoter involves the proximal ATF and AP-1 sites

We have previously shown that the rat PCNA (proliferating cell nuclear antigen) gene promoter is responsive to serum stimulation. In this study, the sequence of the promoter responsive to serum stimulation has been localized in the region between nucleotides −70 and +125 relative to the transcription initiation site. This region contains an ATF site (nucleotides −51 to −44) and an AP‐1 site (nucleotides −64 to −58). Mutation at either the ATF or the AP‐1 site reduced the serum responsiveness of the promoter. In gel mobility shift assays, nuclear extracts from serum stimulated cells, compared to those from quiescent cells, exhibit an increasing binding activity toward a promoter related oligonucleotide (−70 to −42) which includes the ATF site and the AP‐1 site. Formation of the DNA:protein complexes requires the simultaneous involvement of ATF and AP‐1 sites as either element can abrogate the complexes in the competition experiment. Both the distance and sequence are essential to complex formation. Moreover, ATF‐1 but not ATF‐2 (or CREB) has been identified as a major component of the complexes in the antibody supershift or interference experiment. The results of this study suggest that ATF‐1 in association with other factors is involved in regulating the serum stimulation of the rat PCNA promoter activity via the proximal ATF and AP‐1 sites.

Identification and characterization of KvgAS, a two-component system in Klebsiella pneumoniae CG43.

A two-component system encoding gene cluster kvgAS that is present only in virulent Klebsiella pneumoniae CG43 was isolated and its sequence determined. RT-PCR and Southern analysis demonstrated that kvgAS is organized as an operon. No apparent effect of a kvgS deletion on bacterial virulence was observed in a mouse peritonitis model. In the presence of paraquat or 2,2-dipyridyl, the activity of kvgAS promoter in the kvgS mutant was found to be reduced to half of the level in the wild-type strain. The data suggest that the KvgAS system is autoregulated and plays a role in countering free radical stresses and sensing iron-limiting conditions.

Prevalence and characteristics of pks genotoxin gene cluster-positive clinical Klebsiella pneumoniae isolates in Taiwan

The pks gene cluster encodes enzymes responsible for the synthesis of colibactin, a genotoxin that has been shown to induce DNA damage and contribute to increased virulence. The present study investigated the prevalence of pks in clinical K. pneumoniae isolates from a national surveillance program in Taiwan, and identified microbiological and molecular factors associated with pks-carriage. The pks gene cluster was detected in 67 (16.7%) of 400 isolates from various specimen types. Multivariate analysis revealed that isolates of K1, K2, K20, and K62 capsular types (pu2009<u20090.001), and those more susceptible to antimicrobial agents (pu2009=u20090.001) were independent factors strongly associated with pks-carriage. Phylogenetic studies on the sequence type (ST) and pulsed-field gel electrophoresis patterns indicated that the pks-positive isolates belong to a clonal group of ST23 in K1, a locally expanding ST65 clone in K2, a ST268-related K20 group, and a highly clonal ST36:K62 group. Carriage of rmpA, iutC, and ybtA, the genes associated with hypervirulence, was significantly higher in the pks-positive isolates than the pks-negative isolates (95.5% vs. 13.2%, pu2009<u20090.001). Further studies to determine the presence of hypervirulent pks-bearing bacterial populations in the flora of community residents and their association with different disease entities may be warranted.