Rearrangement of a large novel Pseudomonas aeruginosa gene island in strains isolated from a patient developing ventilator-associated pneumonia.

Bacterial gene islands add to the genetic repertoire of opportunistic pathogens. Here, we perform comparative analyses of three Pseudomonas aeruginosa strains isolated sequentially over a 3-week period from a patient with ventilator-associated pneumonia (VAP) who received clindamycin and piperacillin-tazobactam as part of their treatment regime. While all three strains appeared to be clonal by standard pulsed-field gel electrophoresis, whole-genome sequencing revealed subtle alterations in the chromosomal organization of the last two strains; specifically, an inversion event within a novel 124-kb gene island (PAGI 12) composed of 137 open reading frames [ORFs]. Predicted ORFs in the island included metabolism and virulence genes. Overexpression of a gene island-borne putative ß-lactamase gene was observed following piperacillin-tazobactam exposure and only in those strains that had undergone the inversion event, indicating altered gene regulation following genomic remodeling. Examination of a separate cohort of 76 patients with VAP for integration at this tRNA(lys) recombination site demonstrated that patients exhibiting evidence of integration at this site had significantly higher 28-day mortality. These findings provide evidence that P. aeruginosa can integrate, rapidly remodel, and express exogenous genes, which likely contributes to its fitness in a clinical setting.

Secretion of Pseudomonas aeruginosa type III cytotoxins is dependent on pseudomonas quinolone signal concentration.

Pseudomonas aeruginosa is an opportunistic pathogen that can, like other bacterial species, exist in antimicrobial resistant sessile biofilms and as free-swimming, planktonic cells. Specific virulence factors are typically associated with each lifestyle and several two component response regulators have been shown to reciprocally regulate transition between biofilm-associated chronic, and free-swimming acute infections. Quorum sensing (QS) signal molecules belonging to the las and rhl systems are known to regulate virulence gene expression by P. aeruginosa. However the impact of a recently described family of novel quorum sensing signals produced by the Pseudomonas Quinolone Signal (PQS) biosynthetic pathway, on the transition between these modes of infection is less clear. Using clonal isolates from a patient developing ventilator-associated pneumonia, we demonstrated that clinical observations were mirrored by an in vitro temporal shift in isolate phenotype from a non-secreting, to a Type III cytotoxin secreting (TTSS) phenotype and further, that this phenotypic change was PQS-dependent. While intracellular type III cytotoxin levels were unaffected by PQS concentration, cytotoxin secretion was dependent on this signal molecule. Elevated PQS concentrations were associated with inhibition of cytotoxin secretion coincident with expression of virulence factors such as elastase and pyoverdin. In contrast, low concentrations or the inability to biosynthesize PQS resulted in a reversal of this phenotype. These data suggest that expression of specific P. aeruginosa virulence factors appears to be reciprocally regulated and that an additional level of PQS-dependent post-translational control, specifically governing type III cytotoxin secretion, exists in this species.

Presence or absence of lipopolysaccharide O antigens affects type III secretion by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the major causative agents of mortality and morbidity in hospitalized patients due to a multiplicity of virulence factors associated with both chronic and acute infections. Acute P. aeruginosa infection is primarily mediated by planktonic bacteria expressing the type III secretion system (TTSS), a surface-attached needle-like complex that injects cytotoxins directly into eukaryotic cells, causing cellular damage. Lipopolysaccharide (LPS) is the principal surface-associated virulence factor of P. aeruginosa. This molecule is known to undergo structural modification (primarily alterations in the A- and B-band O antigen) in response to changes in the mode of life (e.g., from biofilm to planktonic). Given that LPS exhibits structural plasticity, we hypothesized that the presence of LPS lacking O antigen would facilitate eukaryotic intoxication and that a correlation between the LPS O-antigen serotype and TTSS-mediated cytotoxicity would exist. Therefore, strain PAO1 (A+ B+ O-antigen serotype) and isogenic mutants with specific O-antigen defects (A+ B-, A- B+, and A- B-) were examined for TTSS expression and cytotoxicity. A strong association existed in vitro between the absence of the large, structured B-band O antigen and increased cytotoxicity of these strains. In vivo, all three LPS mutant strains demonstrated significantly increased lung injury compared to PAO1. Clinical strains lacking the B-band O antigen also demonstrated increased TTSS secretion. These results suggest the existence of a cooperative association between LPS O-antigen structure and the TTSS in both laboratory and clinical isolates of P. aeruginosa.

Metabolism of dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylene dioxybiphenyl-2,2'-dicarboxylate (DDB) by human liver microsomes: characterization of metabolic pathways and of cytochrome P450 isoforms involved.

Metabolic fate of DDB and identification of P450 isozymes involved in the metabolism of DDB were investigated in human liver microsomes. DDB was rapidly metabolized to five different metabolites, and the structures of each metabolite were characterized based on UV, mass, and NMR spectral analyses. The major metabolic pathways of DDB in human liver microsomes were identified as O-demethylation of the carboxymethyl moiety (M4) and demethylenation of the methylenedioxyphenyl group (M2). The intramolecular lactonization between the hydroxyl group at the C6 and carboxymethyl group at the C2' of M2 resulted in the generation of M5, which was either hydrolyzed to its hydrolyzed derivative (M1) or further metabolized to the O-demethylated derivative (M3). The interconversion of M1, M2, and M5 took place nonenzymatically depending on the solvent condition. M5 was predominantly detected at the acidic condition, whereas M1 was preferentially detected at the basic environment. Cytochrome P450 (P450) isoform(s) involved in the metabolism of DDB was identified using several in vitro approaches. Chemical inhibition using isoform-selective P450 inhibitors, correlation of DDB metabolites formation with several isoform-specific P450 activities in a panel of liver microsomes, metabolism by microsomes derived from P450 cDNA-expressed B-lymphoblastoid cells, and immunoinhibition by isoform-specific anti-P450 antibodies collectively indicated that CYP1A2, CYP2C9, and CYP3A4 are responsible for the metabolism of DDB. O-Dealkylation of the carboxymethyl group was preferentially catalyzed by CYP1A2, whereas demethylenation of the methylenedioxyphenyl moiety was catalyzed by CYP3A4 and CYP2C9.

Antitumor activity and nephrotoxicity of a novel platinum complex, [1,3-bis(diphenylphosphino)propane](trans-1-dach)platinum(I I) dinitrate.

We have developed a new class of platinum complex [Pt(trans-l-dach)(1,3-bis(phosphino)propane)] dinitrate (KHPC-001) with potent antitumor activity and low nephrotoxicity, confirmed in vitro and compared in vivo with cisplatin, KHPC-001 or cisplatin was intraperitoneally injected on days 1, 5, and 9 into P388-bearing mice and the antitumor effects were compared. In vitro cytotoxicity, Pt accumulation, and DNA cross-link index were measured in P388 and LLC-PK1 cells after treatment with KHPC-001 or cisplatin. Twenty mg/kg (below one-tenth of LD50) of KHPC-001 had stronger antitumor effects than 2 mg/kg (about one-fifth of LD50) of cisplatin and cured 2 out of 6 mice without any toxicity. While the cytotoxicity of KHPC-001 and cisplatin were similar on P388 mouse leukemia cells, this new compound was much less cytotoxic to a kidney-derived line, LLC-PK1. This lower toxicity on the kidney cells was based on its low accumulation, causing less DNA crosslinking. KHPC-001 is a unique third-generation platinum complex with potent antitumor activity and low nephrotoxicity.