Diclofenac sensitizes multi-drug resistant Acinetobacter baumannii to colistin.

Acinetobacter baumannii causes life-threatening infections that are becoming difficult to treat due to increasing rates of multi-drug resistance (MDR) among clinical isolates. This has led the World Health Organization and the CDC to categorize MDR A. baumannii as a top priority for the research and development of new antibiotics. Colistin is the last-resort antibiotic to treat carbapenem-resistant A. baumannii . Not surprisingly, reintroduction of colistin has resulted in the emergence of colistin-resistant strains. Diclofenac is a nonsteroidal anti-inflammatory drug used to treat pain and inflammation associated with arthritis. In this work, we show that diclofenac sensitizes colistin-resistant A. baumannii clinical strains to colistin, in vitro and in a murine model of pneumonia. Diclofenac also reduced the colistin MIC of Klebsiella pneumoniae and Pseudomonas aeruginosa isolates. Transcriptomic and proteomic analyses revealed an upregulation of oxidative stress-related genes and downregulation of type IV pili induced by the combination treatment. Notably, the concentrations of colistin and diclofenac effective in the murine model were substantially lower than those determined in vitro , implying a stronger synergistic effect in vivo compared to in vitro . A pilA mutant strain, lacking the primary component of the type IV pili, became sensitive to colistin in the absence of diclofenac. This suggest that the downregulation of type IV pili is key for the synergistic activity of these drugs in vivo and indicates that colistin and diclofenac exert an anti-virulence effect. Together, these results suggest that the diclofenac can be repurposed with colistin to treat MDR A. baumannii .

Acinetobacter baumannii ATCC 17978 encodes a microcin system with antimicrobial properties for contact-independent competition.

Acinetobacter baumannii is a multidrug-resistant opportunistic pathogen that persists in the hospital environment and causes various clinical infections, primarily affecting immunocompromised patients. A. baumannii has evolved a wide range of mechanisms to compete with neighbouring bacteria. One such competition strategy depends on small secreted peptides called microcins, which exert antimicrobial effects in a contact-independent manner. Here, we report that A. baumannii ATCC 17978 (AB17978) encodes the class II microcin 17 978 (Mcc17978) with antimicrobial activity against closely related Acinetobacter, and surprisingly, also Escherichia coli strains. We identified the genetic locus encoding the Mcc17978 system in AB17978. Using classical bacterial genetic approaches, we determined that the molecular receptor of Mcc17978 in E. coli is the iron-catecholate transporter Fiu, and in Acinetobacter is Fiu's homolog, PiuA. In bacteria, the Ferric uptake regulator (Fur) positively regulates siderophore systems and microcin systems under iron-deprived environments. We found that the Mcc17978 system is upregulated under low-iron conditions commonly found in the host environment and identified a putative Fur binding site upstream of the mcc17978 gene. When we tested the antimicrobial activity of Mcc17978 under different levels of iron availability, we observed that low iron levels not only triggered transcriptional induction of the microcin, but also led to enhanced microcin activity. Taken together, our findings suggest that A. baumannii may utilize microcins to compete with other microbes for resources during infection.

Type VI secretion system mutations reduced competitive fitness of classical Vibrio cholerae biotype.

The gram-negative bacterium Vibrio cholerae is the causative agent of the diarrhoeal disease cholera and is responsible for seven recorded pandemics. Several factors are postulated to have led to the decline of 6th pandemic classical strains and the rise of El Tor biotype V. cholerae, establishing the current 7th pandemic. We investigated the ability of classical V. cholerae of the 2nd and 6th pandemics to engage their type six secretion system (T6SS) in microbial competition against non-pandemic and 7th pandemic strains. We report that classical V. cholerae underwent sequential mutations in T6SS genetic determinants that initially exposed 2nd pandemic strains to microbial attack by non-pandemic strains and subsequently caused 6th pandemic strains to become vulnerable to El Tor biotype V. cholerae intraspecific competition. The chronology of these T6SS-debilitating mutations agrees with the decline of 6th pandemic classical strains and the emergence of 7th pandemic El Tor V. cholerae.

Reactive oxygen species generated in chloroplasts contribute to tobacco leaf infection by the necrotrophic fungus Botrytis cinerea.

Reactive oxygen species (ROS) play fundamental roles in plant responses to pathogen infection, including modulation of cell death processes and defense-related gene expression. Cell death triggered as part of the hypersensitive response enhances resistance to biotrophic pathogens, but favors the virulence of necrotrophs. Even though the involvement of ROS in the orchestration of defense responses is well established, the relative contribution of specific subcellular ROS sources to plant resistance against microorganisms with different pathogenesis strategies is not completely known. The aim of this work was to investigate the role of chloroplastic ROS in plant defense against a typical necrotrophic fungus, Botrytis cinerea. For this purpose, we used transgenic Nicotiana tabacum (tobacco) lines expressing a plastid-targeted cyanobacterial flavodoxin (pfld lines), which accumulate lower chloroplastic ROS in response to different stresses. Tissue damage and fungal growth were significantly reduced in infected leaves of pfld plants, as compared with infected wild-type (WT) counterparts. ROS build-up triggered by Botrytis infection and associated with chloroplasts was significantly decreased (70-80%) in pfld leaves relative to the wild type. Phytoalexin accumulation and expression of pathogenesis-related genes were induced to a lower degree in pfld plants than in WT siblings. The impact of fungal infection on photosynthetic activity was also lower in pfld leaves. The results indicate that chloroplast-generated ROS play a major role in lesion development during Botrytis infection. This work demonstrates that the modulation of chloroplastic ROS levels by the expression of a heterologous antioxidant protein can provide a significant degree of protection against a canonical necrotrophic fungus.

A Burkholderia Type VI Effector Deamidates Rho GTPases to Activate the Pyrin Inflammasome and Trigger Inflammation.

Burkholderia cenocepacia is an opportunistic pathogen of the cystic fibrosis lung that elicits a strong inflammatory response. B. cenocepacia employs a type VI secretion system (T6SS) to survive in macrophages by disarming Rho-type GTPases, causing actin cytoskeletal defects. Here, we identified TecA, a non-VgrG T6SS effector responsible for actin disruption. TecA and other bacterial homologs bear a cysteine protease-like catalytic triad, which inactivates Rho GTPases by deamidating a conserved asparagine in the GTPase switch-I region. RhoA deamidation induces caspase-1 inflammasome activation, which is mediated by the familial Mediterranean fever disease protein Pyrin. In mouse infection, the deamidase activity of TecA is necessary and sufficient for B. cenocepacia-triggered lung inflammation and also protects mice from lethal B. cenocepacia infection. Therefore, Burkholderia TecA is a T6SS effector that modifies a eukaryotic target through an asparagine deamidase activity, which in turn elicits host cell death and inflammation through activation of the Pyrin inflammasome.