Recent data indicate that cystic fibrosis (CF) airway mucus is anaerobic. This suggests that Pseudomonas aeruginosa infection in CF reflects biofilm formation and persistence in an anaerobic environment. P. aeruginosa formed robust anaerobic biofilms, the viability of which requires rhl quorum sensing and nitric oxide (NO) reductase to modulate or prevent accumulation of toxic NO, a byproduct of anaerobic respiration. Proteomic analyses identified an outer membrane protein, OprF, that was upregulated approximately 40-fold under anaerobic versus aerobic conditions. Further, OprF exists in CF mucus, and CF patients raise antisera to OprF. An oprF mutant formed poor anaerobic biofilms, due, in part, to defects in anaerobic respiration. Thus, future investigations of CF pathogenesis and therapy should include a better understanding of anaerobic metabolism and biofilm development by P. aeruginosa.

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Mucoid, mucA mutant Pseudomonas aeruginosa cause chronic lung infections in cystic fibrosis (CF) patients and are refractory to phagocytosis and antibiotics. Here we show that mucoid bacteria perish during anaerobic exposure to 15 mM nitrite (NO2) at pH 6.5, which mimics CF airway mucus. Killing required a pH lower than 7, implicating formation of nitrous acid (HNO2) and NO, that adds NO equivalents to cellular molecules. Eighty-seven percent of CF isolates possessed mucA mutations and were killed by HNO2 (3-log reduction in 4 days). Furthermore, antibiotic-resistant strains determined were also equally sensitive to HNO2. More importantly, HNO2 killed mucoid bacteria (a) in anaerobic biofilms; (b) in vitro in ultrasupernatants of airway secretions derived from explanted CF patient lungs; and (c) in mouse lungs in vivo in a pH-dependent fashion, with no organisms remaining after daily exposure to HNO2 for 16 days. HNO2 at these levels of acidity and NO2 also had no adverse effects on cultured human airway epithelia in vitro. In summary, selective killing by HNO2 may provide novel insights into the important clinical goal of eradicating mucoid P. aeruginosa from the CF airways.

The Polo-like kinases (Plks) are a conserved family of kinases that contribute to cell cycle regulation, particularly in G2 and mitosis. In mammals, there are at least three members of the Plk family. Here we show that Plk3 is a stress response protein that becomes phosphorylated following DNA damage or mitotic spindle disruption. Phosphorylation enhances its kinase activity and is dependent upon ataxia telangiectasiamutated (ATM) in the former case but not the latter. Plk3 associates with complexes of multiple sizes ranging from 150 to greater then 600 kDa. In its unphosphorylated form it elutes from a sizing column at about 400 kDa whereas it associates with complexes of 150 and 600 kDa when phosphorylated. Among the proteins with which it physically associates and utilizes, as substrates are Chk2 and P53. It phosphorylates Chk2 on a residue different from threonine 68 (Thr68), the principal target for ATM. While ATM is necessary for phosphorylation and activation of Chk2 in vivo, Plk3 seems to contribute to its full activation. In its phosphorylated form it also coelutes and forms a complex with unpolymerized tubulin. In aggregate, the data argue that Plk3 is a multifunctional protein that associates with multiple complexes and that contributes to response to stress incurred by DNA damage and mitotic spindle disruption, albeit via different pathways.

Fibroblasts transformed by Fos oncogenes display increased expression of a number of genes implicated in

Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor–induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

Protection from NO gas, a toxic byproduct of anaerobic respiration in Pseudomonas aeruginosa, is mediated by nitric oxide (NO) reductase (NOR), the norCB gene product. Nevertheless, a norCB mutant that accumulated approximately 13.6 microM NO paradoxically survived anaerobic growth. Transcription of genes encoding nitrate and nitrite reductases, the enzymes responsible for NO production, was reduced >50- and 2.5-fold in the norCB mutant. This was due, in part, to a predicted compromise of the [4Fe-4S](2+) cluster in the anaerobic regulator ANR by physiological NO levels, resulting in an inability to bind to its cognate promoter DNA sequences. Remarkably, two O(2)-dependent dioxygenases, homogentisate-1,2-dioxygenase (HmgA) and 4-hydroxyphenylpyruvate dioxygenase (Hpd), were derepressed in the norCB mutant. Electron paramagnetic resonance studies showed that HmgA and Hpd bound NO avidly, and helped protect the norCB mutant in anaerobic biofilms. These data suggest that protection of a P. aeruginosa norCB mutant against anaerobic NO toxicity occurs by both control of NO supply and reassignment of metabolic enzymes to the task of NO sequestration.

Mitosis in human cells is initiated at the end of G2 by activation of the Cdc2/cyclin B complex. Activation occurs by dephosphorylation of the inhibitory residues, threonine 14 (T14) and tyrosine 15 (Y15), on Cdc2 by the Cdc25C phosphatase. Entry into mitosis is regulated by the subcellular relocalization of Cdc2/cyclin B, which is rapidly imported into the nucleus at the end of G2. Here, we show that polo-like kinase 3 (Plk3) is able to phosphorylate Cdc25C primarily on S191, and to a lesser extent on S198 in vitro, both of which are within a nuclear exclusion motif. Following transfection, the S191D Cdc25C mutant leads to an enhanced accumulation of Cdc25C in the nucleus, while the S191A mutant facilitated the Cdc25C nuclear exclusion. Furthermore, translocation of Cdc25C to the nucleus was accompanied by a decrease in Cdc2 phosphorylation on Y15. Plk3-WT overexpression led to a sharp increase in Cdc25C nuclear accumulation, while Plk3-KD overexpression failed to do so. The effect of Plk3 overexpression on Cdc25C was reversed by coexpression of a Plk3 siRNA. These results support a role for the polo kinases in coordinating the translocation and perhaps the timing of both Cdc25C and its target Cdc2/cyclin B to the nucleus upon entry into mitosis.