Extracellular vesicles of Pseudomonas aeruginosa downregulate pyruvate fermentation enzymes and inhibit the initial growth of Staphylococcus aureus

Staphylococcus aureus and Pseudomonas aeruginosa are well-known opportunistic pathogens that frequently coexist in chronic wounds and cystic fibrosis. The exoproducts of P. aeruginosa have been shown to affect the growth and pathogenicity of S. aureus, but the detailed mechanisms are not well understood. In this study, we investigated the effect of extracellular vesicles from P. aeruginosa (PaEVs) on the growth of S. aureus. We found that PaEVs inhibited the S. aureus growth independently of iron chelation and showed no bactericidal activity. This growth inhibitory effect was also observed with methicillin-resistant S. aureus but not with Acinetobacter baumannii, Enterococcus faecalis, S. Typhimurium, E. coli, Listeria monocytogenes, or Candida albicans, suggesting that the growth inhibitory effect of PaEVs is highly specific for S. aureus. To better understand the detailed mechanism, the difference in protein production of S. aureus between PaEV-treated and non-treated groups was further analyzed. The results revealed that lactate dehydrogenase 2 and formate acetyltransferase enzymes in the pyruvate fermentation pathway were significantly reduced after PaEV treatment. Likewise, the expression of ldh2 gene for lactate dehydrogenase 2 and pflB gene for formate acetyltransferase in S. aureus was reduced by PaEV treatment. In addition, this inhibitory effect of PaEVs was abolished by supplementation with pyruvate or oxygen. These results suggest that PaEVs inhibit the growth of S. aureus by suppressing the pyruvate fermentation pathway. This study reported a mechanism of PaEVs in inhibiting S. aureus growth which may be important for better management of S. aureus and P. aeruginosa co-infections

Differential proteomic analysis and pathogenic effects of outer membrane vesicles derived from Acinetobacter baumannii under normoxia and hypoxia

Acinetobacter baumannii is a major causative agent of nosocomial infections and its outer membrane vesicles (AbOMVs) have been shown to be involved in pathogenicity by transporting virulence factors and transferring information for communication between pathogens and host cells. Despite the fact that the infected sites of A. baumannii such as lungs and skin soft tissues are hypoxic, most studies on AbOMV virulence have used AbOMVs prepared under aerobic conditions. The present study aims to elucidate the protein profile and pathogenic impact of AbOMVs released under hypoxic condition. AbOMVs were isolated from A. baumannii under normoxic and hypoxic conditions, and their protein profiles were compared. The different effects of both normoxic and hypoxic AbOMVs in cytokine response from mouse macrophages, cytotoxicity to the human lung epithelial cells, and bacterial invasion were then investigated. Our results showed that A. baumannii under hypoxia released larger amounts of OMVs with different protein profiles. Although the cytotoxic effect of AbOMVs from normoxia and hypoxia were comparable, AbOMVs from normoxia induced higher TNF-α production and invasion of Staphylococcus aureus and Pseudomonas aeruginosa than those from hypoxia. On the other hand, AbOMVs significantly enhanced A. baumannii invasion into lung epithelial cells in a dose-dependent manner. These results clearly demonstrate that AbOMVs released from normoxic and hypoxic have different impacts in pathogenesis. This finding provides new insight into the complex interactions between A. baumannii, coinfecting pathogens and host cells via OMVs, in particular the different pathogenic effects of AbOMVs under normoxic and hypoxic conditions

Selective cytotoxicity of the anti-diabetic drug, metformin, in glucose-deprived chicken DT40 cells

<div><p>Metformin is a biguanide drug that is widely used in the treatment of diabetes. Epidemiological studies have indicated that metformin exhibits anti-cancer activity. However, the molecular mechanisms underlying this activity currently remain unclear. We hypothesized that metformin is cytotoxic in a tumor-specific environment such as glucose deprivation and/or low oxygen (O₂) tension. We herein demonstrated that metformin was highly cytotoxic under glucose-depleted, but not hypoxic (2% O₂) conditions. In order to elucidate the underlying mechanisms of this selective cytotoxicity, we treated exposed DNA repair-deficient chicken DT40 cells with metformin under glucose-depleted conditions and measured cellular sensitivity. Under glucose-depleted conditions, metformin specifically killed <i>fancc</i> and <i>fancl</i> cells that were deficient in FANCC and FANCL proteins, respectively, which are involved in DNA interstrand cross-link repair. An analysis of chromosomal aberrations in mitotic chromosome spreads revealed that a clinically relevant concentration of metformin induced DNA double-strand breaks (DSBs) in <i>fancc</i> and <i>fancl</i> cells under glucose-depleted conditions. In summary, metformin induced DNA damage under glucose-depleted conditions and selectively killed cells. This metformin-mediated selective toxicity may suppress the growth of malignant tumors that are intrinsically deprived of glucose.</p></div

FA pathway-related proteins involved in removing DNA lesions induced by metformin.

<p>(A) Histograms of the IC₅₀ values of metformin in wild-type cells and cell lines deficient in various FANC-related proteins. Cells were treated with metformin under glucose-depleted conditions for 24 h and colonies formed on complete media. All data represent IC₅₀ values ± 95% confidence intervals; (B) The toxicity of metformin to cells deficient in the FANCC or FANCL protein and deficient cell lines stably expressing the indicated transgenes. Data represent the mean ± S.D.; (C) The toxicity of metformin to cells deficient in the TDP1 or PARP1 protein and cells simultaneously deficient in both TDP1 and PARP1 proteins. Data represent the means ±S.D.</p

Toxicity of metformin and comparison of cellular sensitivities to metformin among various DNA repair-deficient DT40 cell lines under glucose-depleted conditions.

<p>(A) Wild-type cells were treated with the indicated doses of metformin for 24 h in complete medium, no glucose medium, or the 2% O₂ hypoxic condition with complete medium, and colonies formed on methylcellulose-containing complete media under normal conditions for 7 days. All data represent the mean ± S.D. normalized to cells not treated with metformin from three independent experiments. In each experiment, relative viabilities were measured as N/N₀ where N is the mean number of colonies at each dose of metformin in treated cells and N₀ is the mean number of colonies in untreated controls; (B) Histograms of the IC₅₀ values of metformin in the wild-type and various DNA repair-deficient cell lines. Cells were treated with metformin under glucose-depleted conditions for 24 h and colonies formed on complete media. All data represent IC₅₀ values ± 95% confidence intervals normalized to cells not treated with metformin from three independent experiments. In each experiment, relative viabilities were measured as N/N₀ where N is the mean number of colonies at each dose in metformin-treated cells and N₀ is the mean number of colonies in untreated controls. Abbreviations: Wt, wild type; NER, nucleotide excision repair; BER, base excision repair; Topo-DNA, repair of DNA-topoisomerase (Topo) crosslinks; TLS, translation DNA synthesis; NHEJ, non-homologous end joining; HR, homologous recombination repair; FA, FA pathway (ICL repair).</p

Induction of chromosomal breakages by metformin under glucose-depleted conditions.

<p>(A) Wild-type cells, cell lines deficient in FANCC or FANCL, and reconstituted cells were incubated with or without 13 μM metformin for 24 h under glucose-depleted conditions; (B) Wild-type cells and cell lines deficient in FANCC or FANCL were incubated with the indicated doses of metformin for 24 h in glucose-depleted or -containing medium. We analyzed 50 metaphase nuclei, and quantified the number of chromosomal aberrations per cell (Y-axis). Data represent the mean ± S.E. Asterisks (*) indicate <i>p</i> < 0.05 by a multiple comparison one-way ANOVA (Tukey’s test). N.S.: not significant (<i>p</i> ≥ 0.05).</p