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Colibactin is a nonribosomal peptide-polyketide synthesized by multi-enzyme complexes encoded by the pks gene cluster. Colibactin-producing Escherichia coli have been demonstrated to induce host DNA damage and promote colorectal cancer (CRC) development. In Taiwan, the occurrence of pyogenic liver abscess (PLA) has been suggested to correlate with an increasing risk of CRC, and Klebsiella pneumoniae is the predominant PLA pathogen in Taiwan. At the asn tRNA loci of the newly sequenced K. pneumoniae 1084 genome, we identified a 208-kb genomic island, KPHPI208, of which a module identical to the E. coli pks colibactin gene cluster was recognized. KPHPI208 consists of eight modules, including the colibactin module and the modules predicted to be involved in integration, conjugation, yersiniabactin production, microcin production, and unknown functions. Transient infection of BALB/c normal liver cells with K. pneumoniae 1084 increased the phosphorylation of histone H2AX, indicating the induction of host DNA damage. Colibactin was required for the genotoxicity of K. pneumoniae 1084, as it was diminished by deletion of clbA gene and restored to the wild type level by trans-complementation with a clbA coding plasmid. Besides, BALB/c mice infected with K. pneumoniae 1084 exhibited enhanced DNA damage in the liver parenchymal cells when compared to the isogenic clbA deletion mutant. By PCR detection, the prevalence of pks-positive K. pneumoniae in Taiwan is 25.6%, which is higher than that reported in Europe (3.5%), and is significantly correlated with K1 type, which predominantly accounted for PLA in Taiwan. Our knowledge regarding how bacteria contribute to carcinogenesis has just begun. The identification of genotoxic K. pneumoniae and its genetic components will facilitate future studies to elucidate the molecular basis underlying the link between K. pneumoniae, PLA, and CRC.

is the most common pathogen of community-acquired meningitis in Taiwan. However, the lack of a physiologically relevant meningitis model for has impeded research into its pathogenesis mechanism. Based on the core genome MLST analyses, the hypervirulent K1 strains, which are etiologically implicated in adult meningitis, mostly belong to a single clonal complex, CC23. Some K1 CC23 strains carry a gene cluster responsible for colibactin production. Colibactin is a small genotoxic molecule biosynthesized by an NRPS-PKS complex, which is encoded by genes located on the island. Compared to other hypervirulent which primarily infect the liver, the colibactin-producing ( ) K1 CC23 strains had significant tropism toward the brain of BALB/c mice. We aimed in this study to develop a physiologically relevant meningitis model with the use of K1 CC23 . Acute meningitis was successfully induced in adult BALB/c male mice through orogastric, intranasal, and intravenous inoculation of K1 CC23 . Besides the typical symptoms of bacterial meningitis, severe DNA damages, and caspase 3-independent cell death were elicited by the colibactin-producing K1 CC23 strain. The deletion of , which abolished the production of colibactin, substantially hindered hypervirulence in the key pathogenic steps toward the development of meningitis. Our findings collectively demonstrated that colibactin was necessary but not sufficient for the meningeal tropism of K1 CC23 , and the mouse model established in this study can be applied to identify other virulence factors participating in the development of this life-threatening disease.

Background Klebsiella pneumoniae liver abscess (KLA) is an invasive disease, and the occurrence of infection is related to its virulence factors and colonization of the host's gastrointestinal (GI) tract. Some animal‐sourced isolates share virulence factors with human pathogens. However, the potential of K. pneumoniae as a zoonotic agent has not been confirmed in murine infection model. Objectives To identify the prevalence and virulence profiles of K. pneumoniae colonization in companion and wild animals and subsequently determine the pathogenicity of selected strains. Methods Forty‐five K. pneumoniae isolates (45/302) were obtained from faeces of companion or wild animals. Virulence factors, gyrA polymerase chain reaction with the restriction fragment length polymorphism (PCR‐RFLP) and pulsed field gel electrophoresis (PFGE) were detected and compared with our previous collection of 60 human pathogens. For KLA model and cytotoxicity test, three animal‐sourced isolates, CHKP0009 (snake, K1, KpII), CHKP0021 (turtle, K2, pLVPK, KpI, cluster I) and CHKP1027 (dog, non‐K1/K2, HV, KpI, cluster III), with similar genotype and/or phenotype to human pathogens were selected and evaluated for their virulence with human hypervirulent K. pneumoniae (hvKp) CG43S. Results The prevalence of K. pneumoniae was higher in companion than wild animals. K. pneumoniae was primarily isolated from dogs, turtles and snakes. Some animal‐sourced isolates carried virulence factors and revealed phylogenetic relatedness with human pathogens. In KLA model, BALB/c mice infected with snake isolate CHKP0009 and dog isolate CHKP1027 survived for 14 days but showed significant bacterial loads in the liver and spleen. Notably, the pet turtle isolate CHKP0021 presented comparable virulence with human hvKp CG43S and induced liver abscess formation. All three selected animal‐sourced isolates could colonize in the GI tract and possess cytotoxic ability. These findings demonstrated pathogenicity of the animal K. pneumoniae isolates. In addition, the high prevalence of K. pneumoniae in companion animals and some isolates with virulence profiles suggested animal‐sourced K. pneumoniae has the zoonotic potential to cause human disease. Conclusion Animals are the natural hosts of zoonotic pathogens. Some animal‐sourced K. pneumoniae isolates are not only pathogenic in vivo but also exhibit phylogenetic relatedness to human pathogens, suggesting the existence of a zoonotic risk for K. pneumoniae between these two populations. The higher prevalence of K. pneumoniae was observed in companion than in wild animals. The predominant animals were dogs, turtles and snakes. Some animal‐sourced isolates carried virulence factors and revealed phylogenetic relatedness with human pathogens, suggesting a zoonotic risk between these two populations. The pathogenicity of selected animal isolates was confirmed in a murine liver abscess model. Companion animals spend more time in contact with humans, and they may serve as reservoirs for opportunistic pathogens that cause human disease.

Gastric cancer (GC) is among the most treatment-refractory epithelial malignancies. Aberrant activation of Wnt/β-catenin-signaling has been implicated in a variety of human cancers, including gastric cancer. Here we report that the elevated expression of lymphoid enhancer binding factor 1 (Lef1) is associated with the TNM (tumor– node–metastasis) stage of gastric cancer. Subsequently, 2,4-diamino-quinazoline (2,4-DAQ), a selective inhibitor of Lef1, was identified to suppress the expression of Wnt/β-catenin target genes such as AXIN2, MYC and LGR5 and result in the suppression of gastric cancer cell growth through the apoptotic pathway. The 2,4-DAQ also exhibited an inhibitory effect on the migration/invasion of gastric cancer cells. Importantly, the treatment of human gastric tumor xenograft with 2,4-DAQ suppressed tumor growth in a nude mouse model. Furthermore, 2,4-DAQ appears effective on patient-derived organoids (PDOs). Transcriptome sequencing analysis also revealed that 2,4-DAQ are more effective on the gastric cancers that exhibit higher expression levels of Wnt-signaling pathway-related genes than their adjacent normal gastric tissues.

Arecoline, the major alkaloid of areca nut, has been shown to cause strong genotoxicity and is considered a potential carcinogen. However, the detailed mechanism for arecoline‐induced carcinogenesis remains obscure. In this study, we noticed that the levels of p21 and p27 increased in two oral squamous cell carcinoma cell lines with high confluence. Furthermore, when treated with arecoline, elevated levels of p21 and p27 could be downregulated through the reactive oxygen species/mTOR complex 1 (ROS/mTORC1) pathway. Although arecoline decreased the activity of mTORC1, the amounts of autophagosome‐like vacuoles or type II LC3 remained unchanged, suggesting that the downregulation of p21 and p27 was independent of autophagy‐mediated protein destruction. Arecoline also caused DNA damage through ROS, indicating that the reduced levels of p21 and p27 might facilitate G 1/S transition of the cell cycle and subsequently lead to error‐prone DNA replication. In conclusion, these data have provided a possible mechanism for arecoline‐induced carcinogenesis in subcytolytic doses in vivo. (Cancer Sci 2012; 103: 1221–1229)

Klebsiella pneumoniae is responsible for a wide range of clinical symptoms. How this bacterium adapts itself to ever-changing host milieu is still a mystery. Recently, small non-coding RNAs (sRNAs) have received considerable attention for their functions in fine-tuning gene expression at a post-transcriptional level to promote bacterial adaptation. Here we demonstrate that Hfq, an RNA-binding protein, which facilitates interactions between sRNAs and their mRNA targets, is critical for K. pneumoniae virulence. A K. pneumoniae mutant lacking hfq (Δhfq) failed to disseminate into extra-intestinal organs and was attenuated on induction of a systemic infection in a mouse model. The absence of Hfq was associated with alteration in composition of envelope proteins, increased production of capsular polysaccharides, and decreased resistance to H(2)O(2), heat shock, and UV irradiation. Microarray-based transcriptome analyses revealed that 897 genes involved in numerous cellular processes were deregulated in the Δhfq strain. Interestingly, Hfq appeared to govern expression of many genes indirectly by affecting sigma factor RpoS and RpoE, since 19.5% (175/897) and 17.3% (155/897) of Hfq-dependent genes belong to the RpoE- and RpoS-regulon, respectively. These results indicate that Hfq regulates global gene expression at multiple levels to modulate the physiological fitness and virulence potential of K. pneumoniae.

The aberrant activation of Wnt signaling has been implicated in a variety of human cancers, including gastric cancer. Given the current hypothesis that cancer arises from cancer stem cells (CSCs), targeting the critical signaling pathways that support CSC self-renewal appears to be a useful approach for cancer therapy. Cell cycle and apoptosis regulator 1 (CCAR1) is a transcriptional coactivator which has been shown to be a component of Wnt/β-catenin signaling, and which plays an important role in transcriptional regulation by β-catenin. However, the function and clinical significance of CCAR1 in gastric cancer have not been elucidated. Here, we show that elevated CCAR1 nuclear expression correlates with the occurrence of gastric cancer. In addition, RNAi-mediated CCAR1 reduction not only suppressed the cell growth and increased apoptosis in AGS and MKN28 cells, but also reduced the migration and invasion ability of these cells. Furthermore, an in vivo xenograft assay revealed that the expression level of CCAR1 was critical for tumorigenesis. Our data demonstrates that CCAR1 contributes to carcinogenesis in gastric cancer and is required for the survival of gastric cancer cells. Moreover, CCAR1 may serve as a diagnostic marker and a potential therapeutic target.

Klebsiella pneumoniae displaying the hypermucoviscosity (HV) phenotype are considered more virulent than HV-negative strains. Nevertheless, the emergence of tissue-abscesses-associated HV-negative isolates motivated us to re-evaluate the role of HV-phenotype. Instead of genetically manipulating the HV-phenotype of K. pneumoniae, we selected two clinically isolated K1 strains, 1112 (HV-positive) and 1084 (HV-negative), to avoid possible interference from defects in the capsule. These well-encapsulated strains with similar genetic backgrounds were used for comparative analysis of bacterial virulence in a pneumoniae or a liver abscess model generated in either naïve or diabetic mice. In the pneumonia model, the HV-positive strain 1112 proliferated to higher loads in the lungs and blood of naïve mice, but was less prone to disseminate into the blood of diabetic mice compared to the HV-negative strain 1084. In the liver abscess model, 1084 was as potent as 1112 in inducing liver abscesses in both the naïve and diabetic mice. The 1084-infected diabetic mice were more inclined to develop bacteremia and had a higher mortality rate than those infected by 1112. A mini-Tn5 mutant of 1112, isolated due to its loss of HV-phenotype, was avirulent to mice. These results indicate that the HV-phenotype is required for the virulence of the clinically isolated HV-positive strain 1112. The superior ability of the HV-negative stain 1084 over 1112 to cause bacteremia in diabetic mice suggests that factors other than the HV phenotype were required for the systemic dissemination of K. pneumoniae in an immunocompromised setting.

Klebsiella pneumoniae-caused liver abscess (KLA) has become a health problem in Taiwan and is continually reported in other countries. Diabetes mellitus, the most common metabolic disorder, underlies half of the KLA patients in Taiwan. The clinical impact of KLA has been well-documented. Nevertheless, the molecular basis regarding how K. pneumoniae causes liver infection, particularly in diabetic individuals, remains unclear. Auto-bioluminescence-expressing K. pneumoniae was inoculated into diabetic mice and age-match naïve control. With the use of in vivo imaging system, translocation of the bioluminescence-expressing K. pneumoniae from intestine to extraintestinal organs, mainly the liver, was noted in 80% of the diabetic mice, whereas the same bacteria causes extraintestinal infections in only 31% of naïve mice. Besides increased morbidity, the severity of hepatic tissue injury was also enhanced in the K. pneumoniae-infected diabetic mice. Upon K. pneumoniae infection, IFN-γ production was significantly evoked in the liver. To mediate IFN-γ signal, STAT (signal transducers and activators of transcription) 1 and 3 were activated in hepatocytes, and so was the expression of IRF (interferon regulatory factor)-1. Moreover, accumulation of neutrophils which was triggered by prolonged production of IL-1β and MIP-2, and significant increases in the level of active caspase 3 and phospho-eIF2α, were exclusively revealed in the K. pneumoniae-infected diabetic mice. The activation of IFN-γ/STAT/IRF-1 signaling demonstrated by this work emphasizes the role of IFN-γ for mediating the hepatic response to K. pneumoniae infection.

Background The effective therapies for oral cancer patients of stage III and IV are generally surgical excision and radiation combined with adjuvant chemotherapy using 5-Fu and Cisplatin. However, the five-year survival rate is still less than 30% in Taiwan. Therefore, evaluation of effective drugs for oral cancer treatment is an important issue. Many studies indicated that aurora kinases (A, B and C) were potential targets for cancer therapies. Reversine was proved to be a novel aurora kinases inhibitor with lower toxicity recently. In this study, the potentiality for reversine as an anticancer agent in oral squamous cell carcinoma (OSCC) was evaluated. Methods Effects of reversine on cell growth, cell cycle progress, apoptosis, and autophagy were evaluated mainly by cell counting, flow cytometry, immunoblot, and immunofluorescence. Results The results demonstrated that reversine significantly suppressed the proliferation of two OSCC cell lines (OC2 and OCSL) and markedly rendered cell cycle arrest at G2/M stage. Reversine also induced cell death via both caspase-dependent and -independent apoptosis. In addition, reversine could inhibit Akt/mTORC1 signaling pathway, accounting for its ability to induce autophagy. Conclusions Taken together, reversine suppresses growth of OSCC via multiple mechanisms, which may be a unique advantage for developing novel therapeutic regimens for treatment of oral cancer in the future.