Acyl Carrier Protein 3 Is Involved in Oxidative Stress Response in Pseudomonas aeruginosa.

The human opportunistic pathogen Pseudomonas aeruginosa expresses three acyl carrier proteins (ACPs): AcpP, Acp1, and Acp3. The function of AcpP in membrane fatty acid synthesis (FAS) was confirmed recently, but the physiological roles of Acp1 and Acp3 remain unclear. To address this, we investigated the physiological role of Acp3 in P. aeruginosa. We found that expression of Acp3 dramatically increases in the log phase of cell growth and that its transcription is under the control of the QS regulators LasR and RhlR. Deletion of acp3 from P. aeruginosa strain PAO1 results in thicker biofilm formation, increased resistance of the strain to hydrogen peroxide, and higher persistence in a mouse infection model. Tandem affinity purification (TAP) experiments revealed several novel protein-binding partners of Acp3, including KatA, the major catalase in P. aeruginosa. Acp3 was found to repress the catalase activity of KatA and, consistent with inhibition by Acp3, less reactive oxygen species are present in the acp3 deletion strain. Overall, our study reveals that Acp3 has a distinct function from that of the canonical AcpP and may be involved in the oxidative stress response.

p97 Promotes a Conserved Mechanism of Helicase Unloading during DNA Cross-Link Repair.

Interstrand cross-links (ICLs) are extremely toxic DNA lesions that create an impassable roadblock to DNA replication. When a replication fork collides with an ICL, it triggers a damage response that promotes multiple DNA processing events required to excise the cross-link from chromatin and resolve the stalled replication fork. One of the first steps in this process involves displacement of the CMG replicative helicase (comprised of Cdc45, MCM2-7, and GINS), which obstructs the underlying cross-link. Here we report that the p97/Cdc48/VCP segregase plays a critical role in ICL repair by unloading the CMG complex from chromatin. Eviction of the stalled helicase involves K48-linked polyubiquitylation of MCM7, p97-mediated extraction of CMG, and a largely degradation-independent mechanism of MCM7 deubiquitylation. Our results show that ICL repair and replication termination both utilize a similar mechanism to displace the CMG complex from chromatin. However, unlike termination, repair-mediated helicase unloading involves the tumor suppressor protein BRCA1, which acts upstream of MCM7 ubiquitylation and p97 recruitment. Together, these findings indicate that p97 plays a conserved role in dismantling the CMG helicase complex during different cellular events, but that distinct regulatory signals ultimately control when and where unloading takes place.

Antibacterial Diamines Targeting Bacterial Membranes.

Antibiotic resistance is a growing threat to human health exacerbated by a lack of new antibiotics. We now describe a series of substituted diamines that produce rapid bactericidal activity against both Gram-positive and Gram-negative bacteria, including methicillin-resistant Staphylococcus aureus and stationary-phase bacteria. These compounds reduce biofilm formation and promote biofilm dispersal in Pseudomonas aeruginosa. The most potent analogue, 3 (1,13-bis{[(2,2-diphenyl)-1-ethyl]thioureido}-4,10-diazatridecane), primarily acts by depolarization of the cytoplasmic membrane and permeabilization of the bacterial outer membrane. Transmission electron microscopy confirmed that 3 disrupts membrane integrity rapidly. Compound 3 is also synergistic with kanamycin, demonstrated by the checkerboard method and by time-kill kinetic experiments. In human cell toxicity assays, 3 showed limited adverse effects against the HEK293T human kidney embryonic cells and A549 human adenocarcinoma cells. In addition, 3 produced no adverse effects on Caenorhabditis elegans development, survival, and reproduction. Collectively, diamines related to 3 represent a new class of broad-spectrum antibacterials against drug-resistant pathogens.

The role of 2,4-dihydroxyquinoline (DHQ) in Pseudomonas aeruginosa pathogenicity.

Bacteria synchronize group behaviors using quorum sensing, which is advantageous during an infection to thwart immune cell attack and resist deleterious changes in the environment. In Pseudomonas aeruginosa, the Pseudomonas quinolone signal (Pqs) quorum-sensing system is an important component of an interconnected intercellular communication network. Two alkylquinolones, 2-heptyl-4-quinolone (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS), activate transcriptional regulator PqsR to promote the production of quinolone signals and virulence factors. Our work focused on the most abundant quinolone produced from the Pqs system, 2,4-dihydroxyquinoline (DHQ), which was shown previously to sustain pyocyanin production and antifungal activity of P. aeruginosa. However, little is known about how DHQ affects P. aeruginosa pathogenicity. Using C. elegans as a model for P. aeruginosa infection, we found pqs mutants only able to produce DHQ maintained virulence towards the nematodes similar to wild-type. In addition, DHQ-only producing mutants displayed increased colonization of C. elegans and virulence factor production compared to a quinolone-null strain. DHQ also bound to PqsR and activated the transcription of pqs operon. More importantly, high extracellular concentration of DHQ was maintained in both aerobic and anaerobic growth. High levels of DHQ were also detected in the sputum samples of cystic fibrosis patients. Taken together, our findings suggest DHQ may play an important role in sustaining P. aeruginosa pathogenicity under oxygen-limiting conditions.