Redox and Metabolic Regulation of Intestinal Barrier Function and Associated Disorders.

The intestinal epithelium forms a physical barrier assembled by intercellular junctions, preventing luminal pathogens and toxins from crossing it. The integrity of tight junctions is critical for maintaining intestinal health as the breakdown of tight junction proteins leads to various disorders. Redox reactions are closely associated with energy metabolism. Understanding the regulation of tight junctions by cellular metabolism and redox status in cells may lead to the identification of potential targets for therapeutic interventions. In vitro and in vivo models have been utilized in investigating intestinal barrier dysfunction and in particular the free-living soil nematode, Caenorhabditis elegans, may be an important alternative to mammalian models because of its convenience of culture, transparent body for microscopy, short generation time, invariant cell lineage and tractable genetics.

Impaired immune response and barrier function in GSPD-1-deficient C. elegans infected with Klebsiella pneumoniae.

gspd-1-RNAi knockdown Caenorhabditis elegans was used as an immune-compromised model to investigate the role of G6PD in host-pathogen interactions. A shorted lifespan, increased bacterial burden and bacterial translocation were observed in gspd-1-knockdown C. elegans infected with Klebsiella pneumoniae (KP). RNAseq revealed that the innate immune pathway, including clc-1 and tsp-1, was affected by gspd-1 knockdown. qPCR confirmed that tight junction (zoo-1, clc-1) and immune-associated genes (tsp-1) were down-regulated in gspd-1-knockdown C. elegans and following infection with KP. The down-regulation of antimicrobial effector lysozymes, including lys-1, lys-2, lys-7, lys-8, ilys-2 and ilys-3, was found in gspd-1-knockdown C. elegans infected with KP. Deletion of clc-1, tsp-1, lys-7, and daf-2 in gspd-1-knockdown C. elegans infected with KP abolished the shorten lifespan seen in the Mock control. GSPD-1 deficiency in C. elegans resulted in bacterial accumulation and lethality, possibly due to a defective immune response. These findings indicate that GSPD-1 has a protective role in microbial defense in C. elegans by preventing bacterial colonization through bacterial clearance.

Cross-protection induced by highly conserved outer membrane proteins (Omps) in mice immunized with OmpC of Salmonella Typhi or OmpK36 of Klebsiella pneumoniae.

BACKGROUND/PURPOSE: Outer membrane proteins (Omps) are a family of proteins that are highly conserved throughout the evolution of Enterobacteriaceae. Previous studies using sequence comparisons have found a high degree of sequence homology between OmpK36 of Klebsiella pneumoniae and OmpC of Salmonella enterica serovar Typhi. Whether highly conserved OmpC can be directly extrapolated as a common vaccine candidate against K. pneumoniae or other Enterobacteriaceae remains to be verified. METHODS: OmpK36 and OmpC were purified and used to immunize BALB/c mice. After immunization, five mice from each group were injected intraperitoneally with a cell suspension of K. pneumoniae or S. Typhi, and the mice were monitored daily for 14 days to measure the severity of illness and assess their survival. RESULTS: Cross-reacting OmpK36 and OmpC antibodies were identified in the mice immunized with OmpK36 or OmpC. No cross-protection was observed in the mice immunized with OmpC in the presence of K. pneumoniae infection. CONCLUSION: Although a high degree of similarity was observed for the amino acid sequences between OmpK36 and OmpC, our results suggested that no cross-protection occurred in the mice challenged with other species.

Immune response and pathophysiological features of Klebsiella pneumoniae liver abscesses in an animal model.

Capsular serotypes K1 and K2, the rmpA gene (a regulator of the mucoid phenotype) and aerobactin from Klebsiella pneumoniae have been identified as the major virulence factors for pyogenic liver abscesses with high morbidity, mortality and severe complications. The pathological mechanisms remain unclear. In this study, we compared liver immune responses and pathological changes in response to different serotypes of K. pneumoniae infections. A mouse model was used to investigate cytokine and chemokine production, histopathology findings, phagocytic uptake and mortality induced by serotypes K1 (magA(+), rmpA(+), aerobactin(+)), K2 (magA(-), rmpA(+), aerobactin(+)), K62 (magA(-), rmpA(-), aerobactin(-)) and an acapsulated isogenic K1 mutant (ΔK1, magA(+), rmpA(+), aerobactin(+)). K. pneumoniae serotypes K1 and K2 showed lower 50% lethal dose values and more phagocytic resistance to neutrophils than K62 and the ΔK1 mutant. In sequential liver samples, viable bacteria counts increased 3 h to 3 days after low-dose inoculation (<10(1) colony-forming unit (cfu)) with K1 and K2, while K62 and ΔK1 cleared rapidly and became undetectable even with high-dose inoculation (∼2.9 × 10(5) cfu). Time-dependent increases in cytokines and chemokines, including tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, IL-10, keratinocyte-derived chemokines and macrophage inflammatory protein-2, were observed in the serum and liver tissue of K1- and K2-infected mice, and severe disease progression manifesting as microabscesses was also identified. K62 and ΔK1 inoculation did not result in similar immune responses and histological changes. These findings illustrate the critical role of phagocytic resistance against innate immunological defense mechanisms as well as its contribution to the development of liver abscesses.

Klebsiella pneumoniae outer membrane porins OmpK35 and OmpK36 play roles in both antimicrobial resistance and virulence.

OmpK35 and OmpK36 are the major outer membrane porins of Klebsiella pneumoniae. In this study, a virulent clinical isolate was selected to study the role of these two porins in antimicrobial resistance and virulence. The single deletion of ompK36 (ΔompK36) resulted in MIC shifts of cefazolin, cephalothin, and cefoxitin from susceptible to resistant, while the single deletion of ompK35 (ΔompK35) had no significant effect. A double deletion of ompK35 and ompK36 (ΔompK35/36) further increased these MICs to high-level resistance and led to 8- and 16-fold increases in the MICs of meropenem and cefepime, respectively. In contrast to the routine testing medium, which is of high osmolarity, susceptibility tests using low-osmolarity medium showed that the ΔompK35 mutation resulted in a significant (≥ 4-fold) increase in the MICs of cefazolin and ceftazidime, whereas a ΔompK36 deletion conferred a significantly (4-fold) lower increase in the MIC of cefazolin. In the virulence assays, a significant (P < 0.05) defect in the growth rate was found only in the ΔompK35/36 mutant, indicating the effect on metabolic fitness. A significant (P < 0.05) increase in susceptibility to neutrophil phagocytosis was observed in both ΔompK36 and ΔompK35/36 mutants. In a mouse peritonitis model, the ΔompK35 mutant showed no change in virulence, and the ΔompK36 mutant exhibited significantly (P < 0.01) lower virulence, whereas the ΔompK35/36 mutant presented the highest 50% lethal dose of these strains. In conclusion, porin deficiency in K. pneumoniae could increase antimicrobial resistance but decrease virulence at the same time.

Contribution of outer membrane protein K36 to antimicrobial resistance and virulence in Klebsiella pneumoniae.

OBJECTIVES: Loss of outer membrane protein (Omp) is commonly encountered in multidrug-resistant Klebsiella pneumoniae. However, little is known about the association between Omp loss and virulence. In the present study, this association was investigated in K. pneumoniae. METHODS: An OmpK36-deficient mutant (DeltaOmpK36) was derived from a virulent clinical isolate by targeted gene insertion. Antimicrobial susceptibility was tested by microbroth dilution and disc diffusion. Virulence was assessed by serum resistance, phagocytosis, clearance of viable bacteria in the liver and lethality in mice following inoculation with bacteria. RESULTS: Susceptibility tests showed that DeltaOmpK36 contributed to the resistance to cefazolin and cefoxitin but not to resistance to late-generation cephalosporins. In vitro assays demonstrated that loss of OmpK36 decreased the resistance to neutrophil phagocytosis and increased the resistance to serum killing during the first hour of the assay, but did not influence the growth rate when compared with the parental strain. Intraperitoneal injection of similar doses ( approximately 4 x 10(4) cfu) of the parental strain and DeltaOmpK36 led to significantly fewer viable bacteria in the liver 24 h post-inoculation in DeltaOmpK36-inoculated mice. In the mice LD(50) (the bacterial dose that caused 50% death) assay, the parental strain was approximately 100-fold more lethal ( approximately 10(3) cfu) than the DeltaOmpK36 mutant ( approximately 10(5) cfu). CONCLUSIONS: Loss of OmpK36 in K. pneumoniae resulted in increased antimicrobial resistance, increased susceptibility to neutrophil phagocytosis, increased resistance to serum killing and reduced virulence.

Widespread dissemination of aminoglycoside resistance genes armA and rmtB in Klebsiella pneumoniae isolates in Taiwan producing CTX-M-type extended-spectrum beta-lactamases.

Among 235 extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL) isolates collected from a nationwide surveillance performed in Taiwan, 102 (43.4%) were resistant to amikacin. Ninety-two of these 102 (90.2%) isolates were carrying CTX-M-type beta-lactamases individually or concomitantly with SHV-type or CMY-2 beta-lactamases. The armA and rmtB alleles were individually detected in 44 and 37 of these 92 isolates, respectively. One isolate contained both armA and rmtB. The coexistence of the aac(6')-Il and rmtB genes was detected in three isolates. CTX-M-type beta-lactamase genes belonging to either group 1 (CTX-M-3 and CTX-M-15) or group 9 (CTX-M-14) were found in all armA- or rmtB-bearing ESBL-producing K. pneumoniae isolates, and all were conjugatively transferable. All except one of the isolates bearing armA produced CTX-M enzymes of group 1, and the remaining isolate bearing armA produced a group 9 CTX-M-type beta-lactamase. On the contrary, in the majority of rmtB carriers, the CTX-M-type beta-lactamase belonged to group 9 (62.2%). Molecular typing revealed that the amikacin-resistant ESBL-producing K. pneumoniae isolates were epidemiologically unrelated, indicating that the acquisition of resistance was not through the spread of a resistant clone or a resistance plasmid. A tandem repeat or multiple copies of bla(CTX-M-3) were found in some armA-bearing isolates. An ISEcp1 insert was found in all CTX-M ESBL-producing K. pneumoniae isolates carrying armA or rmtB. In conclusion, the concomitant presence of a 16S rRNA methylase gene (armA or rmtB) and bla(CTX-M) among amikacin-resistant ESBL-producing K. pneumoniae isolates is widespread in Taiwan.

Effect in virulence of switching conserved homologous capsular polysaccharide genes from Klebsiella pneumoniae serotype K1 into K20.

The capsular polysaccharides in different serotypes of Klebsiella pneumoniae (KP) coded by the (CPS) gene cluster are characterized by a conserved and a hyper-variable region. We performed a virulence study by switching genes in the highly conserved region of the CPS cluster between strains. Six genes in the CPS conserved region in serotype K20, including galF, acidPPc, wzi, wza, wzb and wzc, were knocked out and replaced by the homologous genes from serotype K1. Compared to the parental K20 strain, the mutants showed a decline in lethality (LD50) in mice from 10-fold to > 105-fold and were categorized in terms of the effect on virulence as low (L) for galF and acidPPC, moderate (M) for wzi, and high (H) for wza, wzb and wzc. Although substituting the acidPPC gene from K1 for acidPPC in the K20 strain fully restored virulence, substitution with the wzi, wza, wzb or wzc homologs from K1 did not. The restoration with wzi from K1 led to a partial restoration of virulence, with the LD50 in mice changing from 104 to 103 CFU. For the wza, wzb and wzc genes, Complementation of K20 wza, wzb and wzc from K1 resulted in varied degrees of lethality in mice. Variable improvement in serum killing and phagocytosis was observed when the knockout mutants were compared with the gene-switched strains. In conclusion, homologous genes for capsule synthesis failed to exhibit the same functionality when switched between serotypes and virulence was decreased in different degree in according to the genes' homology.

A Common Flanking Region in Promiscuous Plasmids Encoding blaNDM-1 in Klebsiella pneumoniae Isolated in Singapore.

Bacteria encoding the New Delhi metallo-ß-lactamase gene (blaNDM-1) are regarded as superbugs for their resistance to multiple antibiotics. Plasmids encoding blaNDM-1 have been observed to be spreading among gram-negative bacteria around the world. Previous studies have demonstrated that multiple modifications of blaNDM-1-harboring plasmids might contribute to the spread of the gene. In this study, we analyzed blaNDM-1-encoding plasmids from two Klebsiella pneumoniae isolates, DU7433 and DU1301, found to be unrelated by pulsed field gel electrophoresis and multilocus sequencing typing (DU7433: ST14 and DU1301: ST11), and compared them with previously published plasmids. Although strains DU1301, DU7433, and previously published strain DU43320 carried unrelated plasmids, their transconjugants exhibited similar antimicrobial resistance profiles. Transconjugants lacked the resistance to aztreonam, ciprofloxacin, gentamicin, tetracycline, and trimethoprim/sulfamethoxazole when compared with the corresponding clinical isolates. Plasmids pTR1 from DU1301 and pTR2 from DU7433 had completely different plasmid backbones except a short conserved region of blaNDM-1 and ble flanked with truncated or nontruncated ISAba125 and trpF. The presence of this common region among known blaNDM-1-carrying plasmids implies that the dissemination of blaNDM-1 may be facilitated by mobilization of this conserved immediate region among different plasmids. Control measures should be strictly enforced whenever increasing incidences of epidemiological unrelated strains were identified.

Quantification and comparison of virulence and characteristics of different variants of carbapenemase-producing Klebsiella pneumoniae clinical isolates from Taiwan and the United States.

BACKGROUND/PURPOSE: The emergence of Klebsiella pneumoniae carbapenemase (KPC)-producing strains is a challenge for clinicians. The characteristics and virulence of variants of KPC-producing K. pneumoniae isolates were evaluated. METHODS: Five clinical isolates-three KPC subtypes from Taiwan (KPC2-TW, KPC3-TW, and KPC17-TW) and two clinical strains from the United States (US; KPC2-US, KPC3-US)-were included. Virulent traits and capsular serotypes were analyzed by Polymerase Chain Reaction (PCR). Serum killing, neutrophil phagocytosis, and mice lethargy studies were performed to evaluate virulence. RESULTS: Multilocus sequence typing (MLST) demonstrated that KPC2-TW and KPC17-TW belonged to sequence type (ST)11, and KPC2-US, KPC3-US, and KPC3-TW to ST258. KPC3-TW expressed capsular serotype K1, whereas the others were non-K1/K2/K5 isolates. MLST analysis indicated that ST11 strains were serum resistant, whereas ST258 isolates were serum sensitive. ST11 isolates exhibited significantly higher 15-minute phagocytic rates than ST258 isolates (70.28 ± 16.68% vs. 34.88 ± 10.52%, p < 0.001). The capsular serotype K1 strain was more resistant to neutrophil phagocytosis than non-K1/K2/K5 isolates (27.1 ± 10.23% vs. 54.46 ± 20.94%, p = 0.050). All KPC-producing strain variants from Taiwan and the US demonstrated less virulence in a mouse lethality study, where the LD50 ranged from approximately 10(6) colony-forming units (CFU) to >10(7) CFU. Immunological responses were not significantly correlated with KPC subtype; however, responses were associated with MLST and capsular serotype. CONCLUSION: Production of KPC itself was not associated with increased virulence despite different variants of KPC. The ST11 KPC-producing strain was resistant to serum killing, whereas capsular ss K1 was associated with resistance to neutrophil phagocytosis.

Andrographolide suppresses endothelial cell apoptosis via activation of phosphatidyl inositol-3-kinase/Akt pathway.

Andrographolide (Andro), an active component isolated from the Chinese official herbal Andrographis paniculata, which has been reported to prevent oxygen radical production and thus prevent inflammatory diseases. In this study, we investigated the molecular mechanisms and signaling pathways by which Andro protects human umbilical vein endothelial cells (HUVECs) from growth factor (GF) deprivation-induced apoptosis. Results demonstrated that HUVECs undergo apoptosis after 18 hr of GF deprivation but that this cell death was suppressed by the addition of Andro in a concentration-dependent manner (1-100 microM). Andro suppresses the mitochondrial pathway of apoptosis by inhibiting release of cytochrome c into the cytoplasm and dissipation of mitochondrial potential (Deltapsi(m)), as a consequence, prevented caspase-3 and -9 activation. Treatment of endothelial cells with Andro-induced activation of the protein kinase Akt, an anti-apoptotic signal, and phosphorylation of BAD, a down-stream target of Akt. Suppression of Akt activity by wortmannin, by LY-294002 and by using a dominant negative Akt mutant abolished the anti-apoptotic effect of Andro. In contrast, the ERK1/2 activities were not affected by Andro. The ERK1/2 inhibitor, PD98059 failed to antagonize the protective effect of Andro. In conclusion, Andro exerts its anti-apoptotic potential via activation of the Akt-BAD pathway in HUVECs and thus may represent a candidate of therapeutic agent for atherosclerosis.

Magnolol induces apoptosis in vascular smooth muscle.

Magnolol, an active component extracted from Magnolia officinalis, has various pharmacological effects, including potent antioxidant activity. In the present study, we investigated the effect of magnolol on apoptosis in rat vascular smooth muscle cells (VSMCs), using terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) and flow cytometric analysis. Magnolol (5-20 micro M) concentration-dependently induced significant VSMC apoptosis, this effect being blocked by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk, 50 micro M). To study the molecular mechanism, the mitochondrial death pathway was examined. Magnolol increased caspase-3 and caspase-9 activities significantly and reduced the mitochondrial potential (Deltapsi(m)). The levels of B-cell leukaemia/lymphoma-2 (Bcl-2), but not those of Bcl-2-associated X protein (Bax) or Bcl-x(L), were down-regulated concentration dependently by magnolol. In an animal model, balloon angioplasty-induced neointima formation was inhibited significantly by magnolol and Bcl-2 protein levels were reduced. Taken together, these results show that magnolol induces apoptosis in VSMCs via the mitochondrial death pathway. This effect is mediated through down-regulation of Bcl-2 protein levels, both in vivo and in vitro. Magnolol thus shows potential as a novel therapeutic agent for the treatment of atherosclerosis and re-stenosis.

Inhibition of peroxisome proliferator-activated receptor gamma prevents the melanogenesis in murine B16/F10 melanoma cells.

The purpose of this study was to investigate if PPARγ plays a role in the melanogenesis. B16/F10 cells were divided into five groups: control, melanin stimulating hormone (α-MSH), α-MSH+retinol, α-MSH+GW9662 (PPARγ antagonist), and GW9662. Cells in the control group were cultured in the Dulbecco's modified Eagle's medium (DMEM) for 48 hrs. To initiate the melanogenesis, cells in all α-MSH groups were cultured in medium containing α-MSH (10 nM) for 48 hrs. Cells were treated simultaneously with retinol (5 µM) in the α-MSH+retinol group. Instead of retinol, GW9662 (10 µM) was cocultured in the α-MSH+GW9662 group. Cells in the final group were cultured in the DMEM with GW9662. All the analyses were carried out 48 hours after treatments. The α-MSH was able to increase cell number, melanin production, and the activity of tyrosinase, the limiting enzyme in melanogenesis. These α-MSH-induced changes were prevented either by retinol or by GW9662. Further analyses of the activities of antioxidant enzymes including glutathione, catalase, and the superoxide dismutase (SOD) showed that α-MSH treatment raised the activity of SOD which was dependent on PPARγ level. According to our results, the α-MSH-induced melanogenesis was PPARγ dependent, which also modulated the expression of SOD.

Different culture medium formulations induce variant protein expression patterns of outer membrane porins in Klebsiella pneumoniae.

Outer membrane porin (OMP) expression has been shown to play an important role in antimicrobial resistance. In this study, we observed that OmpK35 of Klebsiella pneumoniae had varied expression profiles in different nutrient broths. the potential factors that could influence protein expression were assessed. K. pneumoniae (ATCC 13883) was cultured into two commercial available nutrient broths and also into solutions of the individual ingredients. To ensure that OmpK35 was detected, an OmpK35 deficient mutant was generated as control. When OmpK protein expression profiles were analyzed by SDS-PAGE, OmpK35 exhibited two different isoforms. Expression of an additional isoform-like OmpK35 protein was identified in one of the broths. No OmpK35 isoforms were observed when the individual ingredients of beef extract, casein or gelatin were used as culture medium. OmpK35 isoform expression could be repressed by adding more beef extract. In summary, OmpK can exhibit varied protein expression profiles when growing in different nutrient broths. The isoform-like protein expression of OmpK35 may lead to confusion in OmpK protein analysis.

One tube multiplex PCR for simple screening of SCCmec I-V types of methicillin-resistant Staphylococcus aureus.

In this study, a rapid and simple one-tube multiplex PCR (M-PCR) method was developed for Staphylococcal Cassette Chromosome mec (SCCmec), primary screening. One hundred and eight methicillin-resistant Staphylococcus aureus (MRSA) isolates with SCC mec typing results using the Oliveira and de Lencastre method (Oliveira and de Lencastre. Antimicrob. Agents Chemother. 46:2155-2161, 2002), were evaluated for the efficacy of our method. Typing results were also compared to the recently published PCR protocol by Kondo et al., (Kondo et al., Antimicrob Agents Chemother. 51:264-74, 2007). The one-tube M-PCR method showed comparable efficacy to the other two methods. None of the isolates were classified as non-typable in Kondo's and our method. Two formerly assigned SCCmec type I isolates by Oliveira and de Lencastre were determined as SCCmec type IV by Kondo's and the method in this study. In conclusion, the one-tube M-PCR method developed in this study increased the rapidity and simplicity for primary screening SCCmec type I to V MRSA.