Biology subjects

Hinkel, L. A.

Publications and source records attributed to Hinkel, L. A..

4 recordsLinked to original sources

bioRxiv2025

Sphingosylphosphorylcholine (SPC) is a substrate for the Pseudomonas aeruginosa phospholipase C/sphingomyelinase, PlcH

Sphingolipids are critical to eukaryotic cell membrane structure and function and play important roles in a variety of host processes that impact infection. Thus, it is not surprising that many pathogens can perturb host sphingolipid homeostasis, often to promote pathogenesis. Pseudomonas aeruginosa is a common opportunistic pathogen that, among many virulence factors, secretes the dual-functioning hemolytic phospholipase C/sphingomyelinase, PlcH. PlcH contributes to P. aeruginosa pathogenesis in several ways and plcH mutants are defective in nearly every infection model, wherein PlcH has been shown to hydrolyze both phosphatidylcholine and sphingomyelin, resulting in inflammation and rupture of host cell membranes. Here, we demonstrate that PlcH can also hydrolyze sphingosylphosphocholine (SPC, also known as lysosphingomyelin), an important host signaling sphingolipid responsible for regulating cellular and tissue responses such as inflammation and endothelial barrier function. PlcH hydrolyzes sphingomyelin to generate phosphocholine and ceramide, and analogously, here we demonstrate that PlcH hydrolyzes SPC to sphingosine and putatively, phosphocholine. We provide evidence that SPC induction of PlcH is primarily regulated by the sphingosine-responsive SphR regulator and that resultant sphingosine liberated from SPC induces transcription from the other genes in the SphR regulon. This work introduces another way that P. aeruginosa can alter the host sphingolipidome, potentially a different mechanism to promote pathogenesis. The capacity for the hemolytic Clostridium perfringens alpha toxin to also cleave SPC suggests that SPC may be a common substrate for phosphocholine-specific phospholipases C. ImportancePlcH is a secreted phospholipase C/sphingomyelinase that is important for the virulence of P. aeruginosa. Here we show that both P. aeruginosa PlcH and C. perfringens alpha toxin can hydrolyze the signaling phospholipid sphingosylphosphorylcholine (SPC), also called lysosphingomyelin. Thus, SPC should be considered a potential target for such phospholipases during infection whose resulting hydrolysis can induce sphingosine-sensitive genes.

microbiology↗

bioRxiv2024

The Pseudomonas aeruginosa sphBC genes are important for growth in the presence of sphingosine by promoting sphingosine metabolism

Sphingoid bases, including sphingosine, are important components of the antimicrobial barrier at epithelial surfaces where they can cause growth inhibition and killing of susceptible bacteria. Pseudomonas aeruginosa is a common opportunistic pathogen that is less susceptible to sphingosine than many Gram-negative bacteria. Here, we determined that deletion of the sphBCD operon reduced growth in the presence of sphingosine. Using deletion mutants, complementation, and growth assays in P. aeruginosa PAO1, we determined that the sphC and sphB genes, encoding a periplasmic oxidase and periplasmic cytochrome c, respectively, were important for growth on sphingosine, while sphD was dispensable under these conditions. Deletion of sphBCD in P. aeruginosa PA14, P. protegens Pf-5, and P. fluorescens Pf01 also showed reduced growth in the presence of sphingosine. The P. aeruginosa sphBC genes were also important for growth in the presence of two other sphingoid bases, phytosphingosine and sphinganine. In wild-type P. aeruginosa, sphingosine is metabolized to an unknown non-inhibitory product, as sphingosine concentrations drop in the culture. However, in the absence of sphBC, sphingosine accumulates, pointing to SphC and SphB as having a role in sphingosine metabolism. Finally, metabolism of sphingosine by wild-type P. aeruginosa protected susceptible cells from full growth inhibition by sphingosine, pointing to a role for sphingosine metabolism as a public good. This work shows that metabolism of sphingosine by P. aeruginosa presents a novel pathway by which bacteria can alter host-derived sphingolipids, but it remains an open question whether SphB and SphC act directly on sphingosine.

microbiology↗

bioRxiv2023

Sphingosine induction of the Pseudomonas aeruginosa hemolytic phospholipase C/sphingomyelinase, PlcH

The hemolytic phospholipase C, PlcH, is an important virulence factor for Pseudomonas aeruginosa. PlcH preferentially hydrolyzes sphingomyelin and phosphatidylcholine and this hydrolysis activity can drives tissue damage, inflammation, and interferes with the oxidative burst of immune cells. Among other contributors, transcription of plcH was previously shown to be induced by phosphate starvation via PhoB and by the choline metabolite, glycine betaine, via GbdR. Here, we show that sphingosine can induce plcH transcription and resultant secreted PlcH enzyme activity. This induction is dependent on the sphingosine-sensing transcription regulator SphR. The SphR induction of plcH occurs from the promoter for the gene upstream of plcH that encodes the neutral ceramidase, CerN, and transcriptional read-through of the cerN transcription terminator. Evidence for these conclusions come from mutation of the SphR binding site in the cerN promoter, mutation of the cerN terminator, enhancement of cerN termination by adding the rrnB terminator, and RT-PCR showing that the intergenic region between cerN and plcH is made as RNA during sphingosine, but not choline, induction. We also observe that, like glycine betaine induction, sphingosine induction of plcH is under catabolite repression control, which likely explains why such induction was not seen in other studies using sphingosine in rich media. The addition of sphingosine as a novel inducer for PlcH points to regulation of plcH transcription as a site for integration of multiple host-derived signals.

microbiology↗

bioRxiv2021

Creatine utilization as a sole nitrogen source in Pseudomonas putida KT2440 is transcriptionally regulated by CahR

Glutamine amidotransferase-1 domain-containing AraC-family transcriptional regulators (GATRs) are present in the genomes of many bacteria, including all Pseudomonas species. The involvement of several characterized GATRs in amine-containing compound metabolism has been determined, but the full scope of GATR ligands and regulatory networks are still unknown. Here, we characterize Pseudomonas putidas detection of the animal-derived amine compound, creatine, a compound particularly enriched in muscle and ciliated cells by a creatine-specific GATR, PP_3665, here named CahR (Creatine amidohydrolase Regulator). cahR is necessary for transcription of the gene encoding creatinase (PP_3667/creA) in the presence of creatine and is critical for P. putidas ability to utilize creatine as a sole source of nitrogen. The CahR/creatine regulon is small and electrophoretic mobility shift demonstrates strong and specific CahR binding only at the creA promoter, supporting the conclusion that much of the regulon is dependent on downstream metabolites. Phylogenetic analysis of creA orthologs associated with cahR orthologs highlights a strain distribution and organization supporting likely horizontal gene transfer, particularly evident within the genus Acinetobacter. This study identifies and characterizes the GATR that transcriptionally controls P. putida metabolism of creatine, broadening the scope of known GATR ligands and suggesting GATR diversification during evolution of metabolism for aliphatic nitrogen compounds.

microbiology↗