Isolation and characterization of quorum‐sensing signalling molecules in Pseudomonas aeruginosa isolates recovered from nosocomial infections

Pseudomonas aeruginosa is one of the most common pathogens in nosocomial infections. Many studies have documented the role of quorum‐sensing (QS) systems in antibiotic tolerance of P. aeruginosa. N‐acyl homoserine lactones (AHLs) serve as QS signalling molecules and can be a target for modulating bacterial pathogenicity. In this study, nosocomial isolates of P. aeruginosa were characterized for the presence of different types of QS signalling molecules. AHLs were solvent extracted and quantified by determination of β‐galactosidase activity using the Escherichia coli MG4 reporter strain. Further characterization was performed by analytical thin layer chromatography coupled with detection using the Agrobacterium tumefaciens A136 biosensor strain. All P. aeruginosa isolates produced AHLs, but there were differences in the quantity and nature of AHLs. We identified AHLs belonging to C4‐homoserine lactone (HSL), C6‐HSL, C8‐HSL, C10‐HSL and C12‐HSL. AHL profiling of P. aeruginosa isolates showed differences in the amounts and types of AHLs, suggesting differences in the virulence factors and the potential for infection. Our results may be investigated further using animal model systems.     

Recombinant DRB sequences produced by mismatch repair of heteroduplexes during cloning in Escherichia coli

Recombinant chimeric sequences originating from a mixture of the sequences of two different alleles are frequently found after amplification and cloning in Escherichia coli of exon 2 of the major histocompatibility complex (MHC) DRB genes. Several authors have suggested that the recombinant molecules result from in vitro recombination during PCR; nevertheless, a clear experimental demonstration of this hypothesis is lacking. In order to understand the mechanism producing the chimeric sequences, we set up a simple experiment based on the different restriction patterns of parental and recombinant sequences. Our data demonstrate that in the analysed case most of the recombinant variants were not produced by in vitro recombination during PCR, but were the result of the mismatch repair of heteroduplex molecules during cloning in E. coli. The high mutation rate in the α‐helix region of DRB expressed genes, both after cloning in E. coli and after the germ‐line differentiation process in vertebrates, suggests that the observed mutations are the result of similar gene conversion processes, probably favoured by chi‐dependent microrecombination events.     

Expression of toll‐like receptors in T lymphocytes stimulated with N‐(3‐oxododecanoyl)‐L‐homoserine lactone from Pseudomonas aeruginosa

The establishment of chronic Pseudomonas aeruginosa infections is correlated with the disturbance of the host immune system. The P. aeruginosa quorum‐sensing molecule N‐3‐(oxododecanoyl)‐L‐homoserine lactone (3‐O‐C12‐HSL) has the potential to modulate the host immune system. The immune system recognizes pathogens via toll‐like receptors (TLRs). We found that 3‐O‐C12‐HSL induced TLR changes in monocytes. However, the role of T cells in P. aeruginosa infection has not been delineated. In order to understand this activity, we examined whether 3‐O‐C12‐HSL has an effect on the immune function and the expression of TLRs in T lymphocytes. Human peripheral blood mononuclear cells (PBMCs) cells were cultured with 0, 1, 10, 50, or 100 μM 3‐O‐C12‐HSL for 12 h. TLR2/TLR4 expression and T‐lymphocyte proliferation were increased in a dose‐dependent manner, and 100 μM 3‐O‐C12‐HSL significantly increased TLR2 expression. Moreover, tumor necrosis factor‐α production of these PBMCs was inhibited. To conclude, 3‐O‐C12‐HSL can induce lymphocyte cell proliferation. These findings provide a new perspective on our understanding of the persistence of the chronic inflammation that accompanies P. aeruginosa infection.     

Multiple natural killer cell-activating signals are inhibited by major histocompatibility complex class I expression in target cells

Several lines of evidence indicate that major histocompatibility complex class I molecules expressed by target cells can prevent natural killer cell (NK) lysis, possibly by engaging inhibitory receptors expressed by NK cells. On the other hand it is likely that NK cells must be activated to lysis by the recognition of unidentified NK target structures on target cells. To investigate the relationship between positive activation of NK cells by NK target structures versus inhibition by target cell class I molecules, we have examined various NK/target cell interactions for which the expression of inhibitory class I molecules by the target cells is known. The results suggest that specific properties of the target cell other than the absence of class I expression are necessary to activate NK-mediated lysis. Furthermore, different effector cell populations, i.e. freshly isolated versus interleukin-2 activated NK cells, differ in their capacity to kill class I-deficient lymphoblast target cells. In general, class I-deficient target cells that are resistant to direct lysis by a given NK population can be lysed by the NK cells when the reaction is mediated by antibody-dependent cellular cytotoxicity (ADCC). Most significantly, all types of NK-mediated lysis of lymphoblasts, of tumor cells and of almost any target by ADCC can be inhibited by appropriate class I gene expression in the target cell. These results suggest a model in which lysis by NK cells must be triggered by any one of a set of distinct target cell ligands, but that all of these signals can be overruled by class I-mediated inhibition.     

Reduced Numbers of Mature Medullary Thymic Epithelial Cells in SKG Mice

Attenuated T cell receptor (TCR ) signalling contributes to the susceptibility for autoimmunity as shown via mutants of PTPN 22 and Zap70 genes. We here set out to investigate the effect of an attenuated TCR signal on the composition of the thymic epithelial cell (TEC ) compartment. To that extent, we combined flow cytometry and histology and compared the TEC subpopulations of Zap70 wild type with SKG mutant mice. We found an increased cortical TEC compartment in SKG thymi at the expense of reduced numbers of mature medullary TEC s and a 4.8‐fold reduced medulla area. We also found reduced proportions of CD 69⁺‐activated thymocytes among double‐negative, double‐positive and CD 4⁻CD 8⁺ single‐positive stages, reduced absolute numbers of single‐positive thymocytes, diminished expression of Lta and Ltb by CD 4⁻CD 8⁺ single‐positive thymocytes and a diminished expression of Ccl19, a target gene of the lymphotoxin‐b‐receptor. While the reduced thymocyte numbers together with the attenuated TCR signal explain the diminished expression of lymphotoxins, the latter is required for an AIRE ‐independent expression of tissue‐restricted antigens as well as attracting positively selected thymocytes to the medulla. Our results describe altered TEC compartments in SKG mice that are likely to support the development of autoimmunity.     

SdiA Aids Enterohemorrhagic Escherichia coli Carriage by Cattle Fed a Forage or Grain Diet

Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis and life-threatening complications. The main reservoirs for EHEC are healthy ruminants. We reported that SdiA senses acyl homoserine lactones (AHLs) in the bovine rumen to activate expression of the glutamate acid resistance (gad) genes priming EHEC''s acid resistance before they pass into the acidic abomasum. Conversely, SdiA represses expression of the locus of enterocyte effacement (LEE) genes, whose expression is not required for bacterial survival in the rumen but is necessary for efficient colonization at the rectoanal junction (RAJ) mucosa. Our previous studies show that SdiA-dependent regulation was necessary for efficient EHEC colonization of cattle fed a grain diet. Here, we compared the SdiA role in EHEC colonization of cattle fed a forage hay diet. We detected AHLs in the rumen of cattle fed a hay diet, and these AHLs activated gad gene expression in an SdiA-dependent manner. The rumen fluid and fecal samples from hay-fed cattle were near neutrality, while the same digesta samples from grain-fed animals were acidic. Cattle fed either grain or hay and challenged with EHEC orally carried the bacteria similarly. EHEC was cleared from the rumen within days and from the RAJ mucosa after approximately one month. In competition trials, where animals were challenged with both wild-type and SdiA deletion mutant bacteria, diet did not affect the outcome that the wild-type strain was better able to persist and colonize. However, the wild-type strain had a greater advantage over the SdiA deletion mutant at the RAJ mucosa among cattle fed the grain diet.     

Escherichia coli inner membrane protein YciB interacts with ZipA that is important for cell division

Escherichia coli propagates by undergoing cycles of lateral elongation, septum formation, and cell fission at the mid‐cell. A large number of genes involved in these processes have been identified, but it is likely that others remain. A deletion mutant of yciB (ΔyciB) is shorter in the cell length compared to wild type and, in contrast, over‐expression of yciB causes elongation of the cell. Furthermore, the septum localization of ZipA, an essential protein of cell division, is disturbed in a ΔyciB mutant. Purified YciB protein directly interacted with ZipA, which might indicate that YciB is involved in the cell envelope synthesis directed by ZipA in a PBP3‐independent manner.     

Hormone‐sensitive lipase in skeletal muscle: regulatory mechanisms

Aim: The enzymatic regulation of intramuscular triacylglycerol (TG) breakdown has until recently not been well understood. Our aim was to elucidate the role of hormone‐sensitive lipase (HSL), which controls TG breakdown in adipose tissue. Methods: Isolated rat muscle as well as exercising humans were studied. Results: The presence of HSL was demonstrated in all muscle fibre types by Western blotting of muscle fibres isolated by collagenase treatment or after freeze‐drying. The content of HSL varies between fibre types, being higher in oxidative than in glycolytic fibres. Analysed under conditions optimal for HSL, neutral lipase activity in muscle can be stimulated by adrenaline as well as by contractions. These increases are abolished by presence of anti‐HSL antibody during analysis. Moreover, immunoprecipitation with affinity‐purified anti‐HSL antibody causes similar reductions in muscle HSL protein concentration and in measured neutral lipase responses to contractions. The immunoreactive HSL in muscle is stimulated by adrenaline via beta‐adrenergic activation of protein kinase A (PKA). From findings in adipocytes it is likely that PKA phosphorylates HSL at residues Ser⁵⁶³, Ser⁶⁵⁹ and Ser⁶⁶⁰. Contraction probably also enhances muscle‐HSL activity by phosphorylation, because the contraction‐induced increase in HSL activity is increased by the protein phosphatase inhibitor okadaic acid and reversed by alkaline phosphatase. A novel signalling pathway in muscle by which HSL activity may be stimulated by protein kinase C (PKC) via extracellular signal regulated kinase (ERK) has been demonstrated. In contrast to previous findings in adipocytes, in muscle activation of ERK is not necessary for stimulation of HSL by adrenaline. However, contraction‐induced HSL activation is mediated by PKC, at least partly via the ERK pathway. In fat cells ERK is known to phosphorylate HSL at Ser⁶⁰⁰. So, phosphorylation of different sites may explain that in muscle the effects of contractions and adrenaline on HSL activity are partially additive. In line with the view that the two stimuli act by different mechanisms, training increases the contraction‐mediated, but diminishes the adrenaline mediated HSL activation in muscle. Conclusion: The existence and regulation of HSL in skeletal muscle indicate a role of HSL in muscle TG metabolism.     

FOXO1 and TCF7L2 genes involved in metastasis and poor prognosis in clear cell renal cell carcinoma

The goal of this study was to identify genes related to the metastasis of clear cell renal cell carcinoma (CRCC). We analyzed copy number alterations in renal tissue specimens of patients with CRCC patients with or without metastasis by using high‐resolution single‐nucleotide polymorphism (SNP) arrays and then integrated these data with gene expression profiling data obtained using oligonucleotide microarrays. The expression levels of target genes were determined by quantitative real‐time RT‐PCR (qRT‐PCR) with an independent tumor set. An analysis of specimens from 23 CRCC cases with SNP arrays revealed that hemizygous deletions at 10q and 13q were found only in cases of metastatic disease. We found the homozygous deletion of TCF7L2 at 10q25.2 in an aggressive case that had hemizygous deletions at 10q. In addition, a qRT‐PCR analysis of TCF7L2 mRNA levels in tumor samples revealed significantly lower levels in patients with metastasis when compared with those without metastasis. FOXO1 was identified as a down‐regulated gene in the minimal overlapping region of the 13q hemizygous deletion in CRCC. Decreased FOXO1 expression was significantly correlated with metastasis and poor survival outcome. Knockdown of FOXO1 inhibited apoptosis after doxorubicin treatment in CRCC cells and reduced the expression of downstream genes involved in cell proliferation (CDKN1B) and survival (BCL2L11). Lower levels of FOXO1 expression were associated with decreased expression of CDKN1B and BCL2L11 in CRCC specimens. We conclude that FOXO1 and TCF7L2 are involved in metastasis and that molecules in these signaling pathways may be targets for diagnostic procedures and therapies for CRCC. © 2010 Wiley‐Liss, Inc.