Biology subjects

Schutz, K.

Publications and source records attributed to Schutz, K..

9 recordsLinked to original sources

bioRxiv2026

A genome-wide screen in Pseudomonas aeruginosa identifies genes impacting production of the hemolytic phospholipase C/sphingomyelinase, PlcH

The secreted phospholipase C/sphingomyelinase, PlcH, is the heat-labile hemolysin of Pseudomonas aeruginosa and one of its important secreted virulence factors. While there are known and suspected genes that impact PlcH production in P. aeruginosa, we sought to identify additional genes by screening the PA14 transposon mutant library to measure extracellular PlcH enzyme activity induced by choline. The library as a whole had a log2-normal distribution of NPPC activity with notable tails that included the genes of interest. These outlier genes included nearly all of those known to be important for PlcH production in response to choline, including those required for choline metabolism, glycine betaine sensing, and secretion through the outer membrane. Interestingly, higher PlcH production was also seen in mutants of the protease associated genes lon, mucD, and clpA, as well as other genes. Additionally, we identified genes impacting baseline levels of PlcH production, which include genes in the dimethylglycine metabolism locus involved in choline metabolism. The high hit rate of known and suspected genes supports the power of this screen and our verification of these genes by clean deletion in strain PA14 confirm the broad importance of these systems across P. aeruginosa, as previous work was confined to strain PAO1. There were many genes identified in this screen that were not individually examined and the complete screen results reported here should allow others to identify intersection of their genes of interest with PlcH production. ImportancePseudomonas aeruginosa is an important opportunistic pathogen that employs multiple independent virulence factors to cause infection, one of which is the hemolytic phospholipase C/sphingomyelinase PlcH. Using a whole genome screen, we identified both known and previously unknown genes contributing to P. aeruginosa PlcH production. Our findings provide insight into the integration of various cellular processes with PlcH production and identify potential genes that may impact the PlcH expression heterogeneity seen in P. aeruginosa clinical isolates.

microbiology↗

bioRxiv2025

Polygenetic Determinants of Azole Resistance, Tolerance, and Heteroresistance in Candida albicans

Azole antifungals are widely used to treat Candida infections, yet therapeutic failures are common. In addition to resistance, fungal populations can exhibit tolerance and heteroresistance, subpopulation-driven drug responses that can contribute to treatment failure. Here, we performed a genome-wide association study (GWAS) on 557 genetically diverse C. albicans isolates to identify genetic loci associated with fluconazole susceptibility, tolerance, and heteroresistance. We uncovered a complex, polygenic architecture underlying all three traits, involving novel loci linked to stress responses, cell cycle, and genome integrity. Notably, canonical resistance genes were absent from our top associations, reflecting either their low frequency or lineage specificity. Functional validation of several candidate genes confirmed distinct genetic determinants for each drug response. Heritability estimates and conditional analyses revealed that susceptibility and heteroresistance are governed by partially overlapping but largely independent loci, with some genetic variants displaying opposing effects on different traits. This limited overlap suggests that each phenotype represents a distinct evolutionary strategy for drug evasion, which may not be simultaneously addressed by a single therapeutic approach. Our findings underscore the polygenic nature of adaptation to antifungal drugs and expand our understanding of drug response mechanisms in C. albicans. SIGNIFICANCEDespite advances in antifungal therapies, treatment failures in Candida albicans infections remain common and poorly understood. Standard clinical diagnostic tools rely on susceptibility testing to predict treatment outcomes, yet many treatment-refractory infections are caused by strains that appear susceptible in laboratory tests. This study provides the first genome-wide dissection of three distinct drug responses - susceptibility, tolerance, and heteroresistance - across a global population of C. albicans isolates. By identifying novel genetic contributors beyond canonical resistance genes, our findings shift the current paradigm toward a more nuanced, polygenic model of antifungal adaptation. These results underscore the importance of integrating tolerance and heteroresistance into clinical and research frameworks and point to new molecular pathways that could be leveraged to enhance antifungal strategies.

microbiology↗

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↗

bioRxiv2023

Reassessing the origins of pathogenicity in Candida auris and relatives through phylogenomic analysis

Emerging fungal pathogens commonly originate from benign or non-pathogenic strains living in the natural environment. Assessing the evolutionary relationships between pathogenic and non-pathogenic species is one approach for tracing the origins of pathogenicity across species. The recently emerged human pathogen, Candida auris belongs to the Candida/Clavispora clade, a diverse group of 45 yeast species including human pathogens and environmental saprobes. C. auris is believed to have originated in the environment and recently transitioned to a human pathogen. We present a phylogenomic analysis of this clade aimed at testing for patterns implicated in the emergence of pathogenicity using an expanded sample of non-pathogenic strains and species. To build a robust framework for investigating these relationships, we developed a whole-genome sequence dataset of 108 isolates representing 18 species, including 4 newly sequenced species and 18 environmentally isolated strains. Our phylogeny, based on 619 orthologous genes, shows environmentally isolated species and strains interspersed with clinically isolated counterparts, rejecting the hypothesis of a single origin of pathogenicity within the lineage containing C. auris and its closest relatives. Our findings highlight the breadth of environments these yeasts inhabit, and imply, concerningly, that known pathogens could just as easily live outside the human body in diverse natural environments. Based on this result, we suggest that surveillance aimed at detecting emerging pathogens should expand to related environmentally-derived fungi with pathogenic potential. AUTHOR SUMMARYThe rapid rise in the number of fungal pathogens over the past few decades has been linked to climate change, globalization, intensive farming practices, and an increase in immunocompromised individuals. Candida auris is an example of a recently emerged fungal pathogen capable of causing severe disease and large outbreaks in vulnerable patient populations. The evolutionary origins of C. auris are poorly understood, however, they are essential to understanding how and when this pathogen emerged. In this study, we investigated relationships between a sample of pathogenic and non-pathogenic strains and species in the Candida/Clavispora clade, a group of 45 yeast species including human pathogens (including C. auris) and environmental saprobes. We used these relationships to test for patterns that might support differing pathogen emergence hypotheses. We found that the relationships between pathogens and non-pathogens suggest many transitions between humans and other environments, rather than a single origin of pathogenicity. It seems plausible that these pathogens, often found in harsh environmental conditions such as seawater, already possessed traits that make them suitable human pathogens, which are perpetuated by increased at-risk patient populations. We should, therefore, be vigilant in our surveillance for clinical isolation of yeasts belonging to this clade from humans.

evolutionary biology↗

bioRxiv2022

Characterizing biofilm interactions between Ralstonia insidiosa and Chryseobacterium gleum

Ralstonia insidiosa and Chryseobacterium gleum are bacterial species commonly found in potable water systems and these two species contribute to the robustness of biofilm formation in a model six-species community from the International Space Station (ISS) potable water system. Here, we set about characterizing the interaction between these two ISS-derived strains and examining the extent to which this interaction extends to other strains and species in these two genera. The enhanced biofilm formation between the ISS strains of R. insidiosa and C. gleum is robust to starting inoculum and temperature, occurs in some but not all tested growth media, and evidence does not support a soluble mediator or co-aggregation mechanism. These findings shed light on the ISS R. insidiosa and C. gleum interaction, though such enhancement is not common between these species based on our examination of other R. insidiosa and C. gleum strains, as well as other species of Ralstonia and Chryseobacterium. Thus, while the findings presented here increase our understanding of the ISS potable water model system, not all our findings are broadly extrapolatable to strains found outside of the ISS. ImportanceBiofilms present in drinking water systems and terminal fixtures are important for human health, pipe corrosion, and water taste. Here we examine the enhanced biofilm of cu-cultures for two very common bacteria from potable water systems, Ralstonia insidiosa and Chryseobacterium gleum. While strains originally isolated on the International Space Station show enhanced dual-species biofilm formation, terrestrial strains do not show the same interaction properties. This study contributes to our understanding of these two species in both dual and mono-culture biofilm formation.

microbiology↗

bioRxiv2022

A metagenomic investigation of spatial and temporal changes in sewage microbiomes across a university campus

Wastewater microbial communities are not static and can vary significantly across time and space, but this variation and the factors driving the observed spatiotemporal variation often remain undetermined. We used a shotgun metagenomic approach to investigate changes in wastewater microbial communities across 17 locations in a sewer network, with samples collected from each location over a 3-week period. Fecal-derived bacteria constituted a relatively small fraction of the taxa found in the collected samples, highlighting the importance of environmental sources to the sewage microbiome. The prokaryotic communities were highly variable in composition depending on the location within the sampling network and this spatial variation was most strongly associated with location-specific differences in sewage pH. However, we also observed substantial temporal variation in the composition of the prokaryotic communities at individual locations. This temporal variation was asynchronous across sampling locations, emphasizing the importance of independently considering both spatial and temporal variation when assessing the wastewater microbiome. The spatiotemporal patterns in viral community composition closely tracked those of the prokaryotic communities, allowing us to putatively identify the bacterial hosts of some of the dominant viruses in these systems. Finally, we found that antibiotic resistance gene profiles also exhibit a high degree of spatiotemporal variability with most of these genes unlikely to be derived from fecal bacteria. Together these results emphasize the dynamic nature of the wastewater microbiome, the challenges associated with studying these systems, and the utility of metagenomic approaches for building a multi-faceted understanding of these microbial communities and their functional attributes. ImportanceSewage systems harbor extensive microbial diversity, including microbes derived from both human and environmental sources. Studies of the sewage microbiome are useful for monitoring public health and the health of our infrastructure, but the sewage microbiome can be highly variable in ways that are often unresolved. We sequenced DNA recovered from wastewater samples collected over a 3-week period at 17 locations in a single sewer system to determine how these communities vary across time and space. Most of the wastewater bacteria, and the antibiotic resistance genes they harbor, were not derived from human feces, but human usage patterns did impact how the amounts and types of bacteria and bacterial genes we found in these systems varied over time. Likewise, the wastewater communities, including both bacteria and their viruses, varied depending on location within the sewage network, highlighting the challenges, and opportunities, in efforts to monitor and understand the sewage microbiome.

microbiology↗

bioRxiv2021

ACVR1 antibodies exacerbate heterotopic ossification in fibrodysplasia ossificans progressiva (FOP) by activating FOP-mutant ACVR1

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder whose most debilitating pathology is progressive and cumulative heterotopic ossification (HO) of skeletal muscles, ligaments, tendons, and fascia. FOP is caused by mutations in the type I BMP receptor gene ACVR1, which enable ACVR1 to utilize its natural antagonist, Activin A, as an agonistic ligand. The physiological relevance of this property is underscored by the fact that HO in FOP is exquisitely dependent on activation of FOP-mutant ACVR1 by Activin A, an effect countered by inhibition of Activin A via monoclonal antibody treatment. Hence, we surmised that ACVR1 antibodies that block activation of ACVR1 by ligand should also inhibit HO in FOP and provide an additional therapeutic option for this condition. Therefore, we generated ACVR1 monoclonal antibodies that block ACVR1s activation by its ligands. Surprisingly, in vivo, these ACVR1 antibodies stimulate HO and activate signaling of FOP-mutant ACVR1. This property is restricted to FOP-mutant ACVR1 and results from ACVR1 antibody-mediated dimerization of ACVR1. Conversely, wild type ACVR1 is inhibited by ACVR1 antibodies. These results uncover an additional novel property of FOP-mutant ACVR1 and indicate that ACVR1 antibodies should not be considered as therapeutics for FOP.

biochemistry↗