Biology subjects

Asfahl, K. L.

Publications and source records attributed to Asfahl, K. L..

7 recordsLinked to original sources

bioRxiv2025

The type VI secretion system governs strain maintenance in a wild mammalian gut microbiome

Bacteria inhabiting the mammalian gut coexist in dense communities where contact-dependent antagonism mechanisms are widespread. The type VI secretion system (T6SS) is an interbacterial toxin delivery pathway prevalent among gut Bacteroidales, yet its function in naturally evolved microbiomes remains poorly defined. Here, we examine the role of the T6SS in Bacteroides within a physiologically relevant gut community derived from wild mice (the WildR microbiome). Using newly developed genetic tools and a strategy for functional replacement of strains within the WildR community, we demonstrate that the WildR isolate B. acidifaciens employs a T6SS to antagonize co-resident Bacteroidales. We also show that loss of T6SS function compromises the long-term maintenance of B. acidifaciens in the community but not its initial colonization, establishing the system as a determinant of strain persistence. The T6SS we identified resides on an integrative and conjugative element (ICE). ICE-seq, a targeted sequencing approach, reveals that the T6SS-ICE is distributed among select Bacteroidales and Muribaculaceae species in the WildR microbiome, between which it appears to be recently exchanged. We also show that transfer of the T6SS-ICE to WildR isolate Phocaeicola vulgatus confers transient colonization benefits in mice, but is linked to eventual population decline. Our findings demonstrate that the T6SS can stabilize the presence of specific strains within a complex, co-evolved gut microbiome, yet its value is context dependent and constrained by the ecological and physiological landscape of the host community.

microbiology↗

bioRxiv2025

Quorum sensing antiactivators constrain Pseudomonas aeruginosa RhlR activity

Pseudomonas aeruginosa, an opportunistic pathogen, uses a cell-cell communication system called quorum sensing (QS) to regulate gene expression in response to population density. P. aeruginosa QS involves, in part, two transcription factors, LasR and RhlR, that respond to N-acyl homoserine lactone (AHL) signals. Two proteins known as "antiactivators," QteE and QslA, attenuate QS by inhibiting LasR, RhlR, or both. While initial characterization of antiactivation has revealed the considerable influence these factors may have on dampening QS, details regarding the individual impacts of these antiactivators on P. aeruginosa QS activity remain scant. Additionally, the effects of antiactivators on RhlR QS activity and in QS systems in isolates or strain lacking LasR have yet to be explored. To investigate how QteE and QslA each modulate LasR or RhlR independently, we combined gene deletion and over-expression analysis of each antiactivator in wild-type P. aeruginosa (PAO1) and two strains with rhl-dominated QS: clinical isolate E90 and PAO {Delta}lasR {Delta}mexT. As measured with a transcriptional reporter, over-expression of qteE or qslA notably reduced RhlR activity in PAO1 and PAO {Delta}lasR {Delta}mexT, but only expression of qteE had a marked effect on RhlR activity in E90. Expression analysis indicates LasR and RhlR repress QteE transcription, but not QslA. By over-expressing qslA in the absence of QteE and vice versa, we demonstrate that QslA activity and corresponding effects on QS phenotypes can be QteE-dependent in some scenarios. Our results reveal a nuanced role for individual antiactivator proteins in affecting the layered P. aeruginosa QS circuitry. ImportanceQuorum sensing (QS) is a cell signaling mechanism that enables populations of Pseudomonas aeruginosa to coordinate group behaviors such as biofilm formation, virulence factor production, and antibiotic tolerance once a critical cell-density threshold is reached. P. aeruginosa employs two "antiactivator" proteins that attenuate QS at low cell densities, dampening QS activation. The specific effects of individual antiactivators on the complex and hierarchically-arranged P. aeruginosa QS systems remain undefined. Here, we use two strains with rewired QS circuits to independently assess the effects of QS antiactivators on each QS circuit. We find that while one antiactivator selectively targets one QS circuit, the other can broadly target both with strong effects on QS activity. This work reveals an additional layer of complexity to counter-regulation of QS signalling and further defines antiactivation as a mechanism P. aeruginosa uses to finely tune QS responses.

microbiology↗

bioRxiv2023

Umbrella toxin particles produced by Streptomyces block mycelial growth of competing species

The Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive. The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species. Here we show that in addition to small molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-EM structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, leading to our naming them umbrella particles. S. coelicolor encodes three umbrella particles with distinct toxin and lectin composition, and supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, S. griseus, we find inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative mycelial growth. Our data show that Streptomyces umbrella particles mediate competition between vegetative mycelia of related species, a function distinct from small molecule antibiotics, which are produced at the onset of reproductive growth and act broadly. Sequence analyses suggest this role of umbrella particles extends beyond Streptomyces, as we find umbrella loci in nearly one-thousand species across Actinobacteria.

microbiology↗

bioRxiv2021

Genetic and transcriptomic characteristics of RhlR-dependent quorum sensing in cystic fibrosis isolates of Pseudomonas aeruginosa

In people with the genetic disease cystic fibrosis (CF), bacterial infections involving the opportunistic pathogen Pseudomonas aeruginosa are a significant cause of morbidity and mortality. P. aeruginosa uses a cell-cell signaling mechanism called quorum sensing (QS) to regulate many virulence functions. One type of QS consists of acyl-homoserine lactone (AHL) signals produced by LuxI-type signal synthases, which bind a cognate LuxR-type transcription factor. In laboratory strains and conditions, P. aeruginosa employs two AHL synthase/receptor pairs arranged in a hierarchy, with the LasI/R system controlling the RhlI/R system and many downstream virulence factors. However, P. aeruginosa isolates with inactivating mutations in lasR are frequently isolated from chronic CF infections. We and others have shown that these isolates frequently use RhlR as the primary QS regulator. RhlR is rarely mutated in CF and environmental settings. We were interested if there were reproducible genetic characteristics of these isolates and if there was a central group of genes regulated by RhlR in all isolates. We examined five isolates and found signatures of adaptation common to CF isolates. We did not identify a common genetic mechanism to explain the switch from Las-to Rhl-dominated QS. We describe a core RhlR regulon encompassing 20 genes encoding 7 products. These results suggest a key group of QS-regulated factors important for pathogenesis of chronic infection, and position RhlR as a target for anti-QS therapeutics. Our work underscores the need to sample a diversity of isolates to understanding QS beyond what has been described in laboratory strains.

microbiology↗

bioRxiv2021

Evolution of the quorum sensing regulon in cooperating populations of Pseudomonas aeruginosa

The bacterium Pseudomonas aeruginosa is an opportunistic pathogen and it thrives in many different saprophytic habitats. In this bacterium acyl-homoserine lactone quorum sensing (QS) can activate expression of over 100 genes, many of which code for extracellular products. P. aeruginosa has become a model for studies of cell-cell communication and coordination of cooperative activities. We hypothesized that long-term growth of bacteria under conditions where only limited QS-controlled functions were required would result in a reduction in the size of the QS-controlled regulon. To test this hypothesis, we grew P. aeruginosa for about 1000 generations in a condition in which expression of QS-activated genes is required for growth. We compared the QS regulons of populations after about 35 generations to those after about 1000 generations in two independent lineages by using quorum quenching and RNA-seq technology. In one evolved lineage the number of QS-activated genes identified was reduced by about 70% and in the other by about 45%. Our results lend important insights about the variations in the number of QS-activated genes reported for different bacterial strains and, more broadly, about the environmental histories of P. aeruginosa.

microbiology↗

bioRxiv2020

Burkholderia thailandensis methylated hydroxy-alkylquinolines: biosynthesis and antimicrobial activity in co-culture experiments

The bacterium Burkholderia thailandensis produces an arsenal of secondary metabolites that have diverse structures and roles in the ecology of this soil-dwelling bacterium. In liquid co-culture experiments, B. thailandensis secretes an antimicrobial that nearly eliminates another soil bacterium, Bacillus subtilis. To identify the antimicrobial, we used a transposon mutagenesis approach. This screen identified antimicrobial-defective mutants with insertions in the hmqA, hmqC and hmqF genes involved in biosynthesis of a family of 2-alkyl-4(1H)-quinolones called 4-hydroxy-3-methyl-2-alkenylquinolines (HMAQs), which are closely related to the Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs). Insertions also occurred in the previously uncharacterized gene BTH_II1576. Results confirm that BTH_II1576 is involved in generating N-oxide derivatives of HMAQs (HMAQ-NO) in B. thailandensis and that HMAQ-NOs are sufficient to eliminate B. subtilis in co-cultures. Moreover, synthetic HMAQ-NO is [~]50-fold more active than HMAQ. Both the methyl group and the length of the carbon side chain account for high activity of HMAQ-NO against B. subtilis. The results provide new information on the biosynthesis and activities of HMAQs and reveal new insight into how these molecules might be important for the ecology of B. thailandensis. IMPORTANCEThe soil bacterium Burkholderia thailandensis produces 2-alkyl-4(1H)-quinolones, mostly methylated 4-hydroxy-alkenylquinolines, a family of relatively unstudied metabolites similar to molecules also synthesized by Pseudomonas aeruginosa. Several of the methylated 4-hydroxy-alkenylquinolines have antimicrobial activity against other species. We show that N-oxidated methyl-alkenylquinolines are particularly antimicrobial and sufficient to kill Bacillus subtilis in co-cultures. We confirmed their biosynthesis requires the previously unstudied protein HmqL. These results provide new information about the biology of 2-alkyl-4(1H)-quinolones, particularly the methylated 4-hydroxy-alkenylquinolines, which are unique to B. thailandensis. This study also has importance for understanding B. thailandensis secondary metabolites and has implications for potential therapeutic development.

microbiology↗

bioRxiv2019

Non-hierarchical, RhlR-regulated acyl-homoserine lactone quorum sensing in a cystic fibrosis isolate of Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of airway infection in cystic fibrosis (CF) patients. P. aeruginosa employs several hierarchically arranged and interconnected quorum sensing (QS) regulatory circuits to produce a battery of virulence factors such as elastase, phenazines, and rhamnolipids. The QS transcription factor LasR sits atop this hierarchy, and activates the transcription of dozens of genes, including that encoding the QS regulator RhlR. Paradoxically, inactivating lasR mutations are frequently observed in isolates from CF patients with chronic P. aeruginosa infections. In contrast, mutations in rhlR are rare. We have recently shown that in CF isolates, the QS circuitry is often \"rewired\" such that RhlR acts in a LasR-independent manner. To begin understanding how QS activity differs in this \"rewired\" background, we characterized QS activation and RhlR-regulated gene expression in P. aeruginosa E90, a LasR-null, RhlR-active chronic infection isolate. In this isolate, RhlR activates the expression of 53 genes in response to increasing cell density. The genes regulated by RhlR include several that encode virulence factors. Some, but not all, of these genes are present in the QS regulon described in the well-studied laboratory strain PAO1. We also demonstrate that E90 produces virulence factors at similar concentrations to that of PAO1. Unlike PAO1, cytotoxicity by E90 in a three-dimensional lung epithelium cell model is also RhlR-regulated. These data illuminate a \"rewired\" LasR-independent RhlR regulon in chronic infection isolates and suggest that RhlR may be a target for therapeutic development in chronic infections.\n\nAUTHOR SUMMARYPseudomonas aeruginosa is a prominent cystic fibrosis (CF) pathogen that uses quorum sensing (QS) to regulate virulence. In laboratory strains, the key QS regulator is LasR. Some isolates from patients with chronic CF infections appear to use an alternate QS circuitry in which another transcriptional regulator, RhlR, mediates QS. We show that a LasR-null CF clinical isolate engages in QS through RhlR and remains capable of inducing cell death in an in vivo-like lung epithelium cell model. Our findings support the notion that LasR-null clinical isolates can engage in RhlR QS and highlight the centrality of RhlR gene regulation in chronic P. aeruginosa infections.

microbiology↗