Pseudomonas aeruginosa Utilizes Host-Derived Itaconate to Redirect Its Metabolism to Promote Biofilm Formation.

The bacterium Pseudomonas aeruginosa is especially pathogenic, often being associated with intractable pneumonia and high mortality. How P. aeruginosa avoids immune clearance and persists in the inflamed human airway remains poorly understood. In this study, we show that P. aeruginosa can exploit the host immune response to maintain infection. Notably, unlike other opportunistic bacteria, we found that P. aeruginosa alters its metabolic and immunostimulatory properties in response to itaconate, an abundant host-derived immunometabolite in the infected lung. Itaconate induces bacterial membrane stress, resulting in downregulation of lipopolysaccharides (LPS) and upregulation of extracellular polysaccharides (EPS). These itaconate-adapted P. aeruginosa accumulate lptD mutations, which favor itaconate assimilation and biofilm formation. EPS, in turn, induces itaconate production by myeloid cells, both in the airway and systemically, skewing the host immune response to one permissive of chronic infection. Thus, the metabolic versatility of P. aeruginosa needs to be taken into account when designing therapies.

Crystal structure of a Pseudomonas malonate decarboxylase holoenzyme hetero-tetramer.

Pseudomonas species and other aerobic bacteria have a biotin-independent malonate decarboxylase that is crucial for their utilization of malonate as the sole carbon and energy source. The malonate decarboxylase holoenzyme contains four subunits, having an acyl-carrier protein (MdcC subunit) with a distinct prosthetic group, as well as decarboxylase (MdcD-MdcE) and acyl-carrier protein transferase (MdcA) catalytic activities. Here we report the crystal structure of a Pseudomonas malonate decarboxylase hetero-tetramer, as well as biochemical and functional studies based on the structural information. We observe a malonate molecule in the active site of MdcA and we also determine the structure of malonate decarboxylase with CoA in the active site of MdcD-MdcE. Both structures provide molecular insights into malonate decarboxylase catalysis. Mutations in the hetero-tetramer interface can abolish holoenzyme formation. Mutations in the hetero-tetramer interface and the active sites can abolish Pseudomonas aeruginosa growth in a defined medium with malonate as the sole carbon source.Some aerobic bacteria contain a biotin-independent malonate decarboxylase (MDC), which allows them to use malonate as the sole carbon source. Here, the authors present the crystal structure of a Pseudomonas MDC and give insights into its catalytic mechanism and function.

Electron-shuttling antibiotics structure bacterial communities by modulating cellular levels of c-di-GMP.

Diverse organisms secrete redox-active antibiotics, which can be used as extracellular electron shuttles by resistant microbes. Shuttle-mediated metabolism can support survival when substrates are available not locally but rather at a distance. Such conditions arise in multicellular communities, where the formation of chemical gradients leads to resource limitation for cells at depth. In the pathogenic bacterium Pseudomonas aeruginosa PA14, antibiotics called phenazines act as oxidants to balance the intracellular redox state of cells in anoxic biofilm subzones. PA14 colony biofilms show a profound morphogenic response to phenazines resulting from electron acceptor-dependent inhibition of ECM production. This effect is reminiscent of the developmental responses of some eukaryotic systems to redox control, but for bacterial systems its mechanistic basis has not been well defined. Here, we identify the regulatory protein RmcA and show that it links redox conditions to PA14 colony morphogenesis by modulating levels of bis-(3',5')-cyclic-dimeric-guanosine (c-di-GMP), a second messenger that stimulates matrix production, in response to phenazine availability. RmcA contains four Per-Arnt-Sim (PAS) domains and domains with the potential to catalyze the synthesis and degradation of c-di-GMP. Our results suggest that phenazine production modulates RmcA activity such that the protein degrades c-di-GMP and thereby inhibits matrix production during oxidizing conditions. RmcA thus forms a mechanistic link between cellular redox sensing and community morphogenesis analogous to the functions performed by PAS-domain-containing regulatory proteins found in complex eukaryotes.

Stimulatory and inhibitory effects of PKC isozymes are mediated by serine/threonine PKC sites of the Cav2.3α1 subunits.

Protein kinase C (PKC) isozymes modulate voltage-gated calcium (Cav) currents through Cav2.2 and Cav2.3 channels by targeting serine/threonine (Ser/Thr) phosphorylation sites of Cavα1 subunits. Stimulatory (Thr-422, Ser-2108 and Ser-2132) and inhibitory (Ser-425) sites were identified in the Cav2.2α1 subunits to PKCs ßII and ε. In the current study, we investigated if the homologous sites of Cav2.3α1 subunits (stimulatory: Thr-365, Ser-1995 and Ser-2011; inhibitory: Ser-369) behaved in similar manner. Several Ala and Asp mutants were constructed in Cav2.3α1 subunits in such a way that the Ser/Thr sites can be examined in isolation. These mutants or WT Cav2.3α1 along with auxiliary ß1b and α2/δ subunits were expressed in Xenopus oocytes and the effects of PKCs ßII and ε studied on the barium current (IBa). Among these sites, stimulatory Thr-365 and Ser-1995 and inhibitory Ser-369 behaved similar to their homologs in Cav2.2α1 subunits. Furthermore PKCs produced neither stimulation nor inhibition when stimulatory Thr-365 or Ser-1995 and inhibitory Ser-369 were present together. However, the PKCs potentiated the IBa when two stimulatory sites, Thr-365 and Ser-1995 were present together, thus overcoming the inhibitory effect of Ser-369. Taken together net PKC effect may be the difference between the responses of the stimulatory and inhibitory sites.

The Pseudomonas aeruginosa efflux pump MexGHI-OpmD transports a natural phenazine that controls gene expression and biofilm development.

Redox-cycling compounds, including endogenously produced phenazine antibiotics, induce expression of the efflux pump MexGHI-OpmD in the opportunistic pathogen Pseudomonas aeruginosa Previous studies of P. aeruginosa virulence, physiology, and biofilm development have focused on the blue phenazine pyocyanin and the yellow phenazine-1-carboxylic acid (PCA). In P. aeruginosa phenazine biosynthesis, conversion of PCA to pyocyanin is presumed to proceed through the intermediate 5-methylphenazine-1-carboxylate (5-Me-PCA), a reactive compound that has eluded detection in most laboratory samples. Here, we apply electrochemical methods to directly detect 5-Me-PCA and find that it is transported by MexGHI-OpmD in P. aeruginosa strain PA14 planktonic and biofilm cells. We also show that 5-Me-PCA is sufficient to fully induce MexGHI-OpmD expression and that it is required for wild-type colony biofilm morphogenesis. These physiological effects are consistent with the high redox potential of 5-Me-PCA, which distinguishes it from other well-studied P. aeruginosa phenazines. Our observations highlight the importance of this compound, which was previously overlooked due to the challenges associated with its detection, in the context of P. aeruginosa gene expression and multicellular behavior. This study constitutes a unique demonstration of efflux-based self-resistance, controlled by a simple circuit, in a Gram-negative pathogen.

Contribution of protein kinase Cα in the stimulation of insulin by the down-regulation of Cavß subunits.

Voltage-gated calcium (Cav) channels and protein kinase C (PKC) isozymes are involved in insulin secretion. In addition, Cavß, one of the auxiliary subunits of Cav channels, also regulates the secretion of insulin as knockout of Cavß3 (ß3(-/-)) subunits in mice led to efficient glucose homeostasis and increased insulin levels. We examined whether other types of Cavß subunits also have similar properties. In this regard, we used small interfering RNA (siRNA) of these subunits (20 µg each) to down-regulate them and examined blood glucose, serum insulin and PKC translocation in isolated pancreatic ß cells of mice. While the down-regulation of Cavß2 and ß3 subunits increased serum insulin levels and caused efficient glucose homeostasis, the down-regulation of Cavß1 and ß4 subunits failed to affect both these parameters. Examination of PKC isozymes in the pancreatic ß-cells of Cavß2- or ß3 siRNA-injected mice showed that three PKC isozymes, viz., PKC α, ßII and θ, translocated to the membrane. This suggests that when present, Cavß2 and ß3 subunits inhibited PKC activation. Among these three isozymes, only PKCα siRNA inhibited insulin and increased glucose concentrations. It is possible that the activation of PKCs ßII and θ is not sufficient for the release of insulin and PKCα is the mediator of insulin secretion under the control of Cavß subunits. Since Cavß subunits are present intracellularly, it is possible that they (1) inhibited the translocation of PKC isozymes to the membrane and (2) decreased the interaction between Cav channels and PKC isozymes and thus the secretion of insulin.