Biology subjects

Carlson, E. E.

Publications and source records attributed to Carlson, E. E..

13 recordsLinked to original sources

bioRxiv2026

Development of binding and activity inhibition assays for the antibiotic resistance-associated protein PhoQ

Antimicrobial resistant infections present a growing threat to public health and were associated with or directly caused 6 million deaths globally in 2021. This huge death toll highlights the need for novel strategies to address AMR infections. Interfering with the regulation of resistance mechanisms could provide an alternative approach to treat drug-resistant infections. PhoQ, a sensor histidine kinase ubiquitous amongst gram-negative bacteria, regulates several virulence factors, as well as resistance to outer membrane-targeting antibiotics, making it an attractive target for adjuvant therapy development. However, the identification of potent small molecule inhibitors is limited by the assays available for in vitro assessment of binding and activity inhibition in PhoQ. Thus, we sought to investigate the use of a fluorescence-based assay to evaluate enzymatic activity, as well as a thermal shift assay to assess inhibitor-protein binding in PhoQ. Together, these newly implemented protocols are valuable contributors to the toolbox of methods available for the development of PhoQ-targeted inhibitors to block this major contributor to antimicrobial resistance.

biochemistry↗

bioRxiv2025

Cobalt and nickel ion synergy promotes gene duplication amplification and enables stable metal resistance in Shewanella oneidensis

Microbial species are integral in environmental homeostasis and have thus developed diverse strategies to adapt, survive, and proliferate under exogenous stress. The growing demand for nanoscale battery materials has led to increasing environmental concentrations of nanomaterials and their constituent metal ions. Previous work has shown that the gram-negative bacterium Shewanella oneidensis is able to rapidly evolve resistance to one such nanomaterial, lithiated nickel manganese cobalt oxide (NMC). Yet, the specific stimuli that trigger resistance evolution and the ensuing genomic changes were previously unknown. Here, we demonstrate that the combination of cobalt and nickel ions released from NMC trigger a gene duplication amplification (GDA) event that enables stable nanomaterial and metal resistance. Amplification copy number is highly dynamic over the period of study, yet amplification events persist over prolonged recovery in the absence of metal stress. Growth rate comparisons reveal no physiological cost associated with high copy GDA. The stability of this genomic mutation combined with the lack of observable fitness cost distinguishes this genomic perturbation from previously reported GDA events. Lastly, the observed GDA event was unique to the combination of cobalt and nickel, which implies that the intracellular targets of these metal ions have a specific interaction that yield resistance. Ultimately, we report a dynamic and specific GDA event that allows S. oneidensis to survive metal stress. This work not only illuminates the broader ecological consequences associated with introducing nanomaterials and metals into the environment but also provides insight into the larger scope of bacterial resistance mechanisms. ImportanceStudying mechanisms of microbial adaptation and resistance evolution is critical for understanding the role of bacteria in the environment and developing new strategies to combat antimicrobial resistance to modern therapeutics. Technological innovation has often led to the introduction of novel environmental stressors that impart pressure on microbes to evolve resistance. This may ultimately lead to novel resistance genes or gene cassettes in the environmental gene pool, which could further promote antimicrobial resistance in pathogenic organisms. In this study, we highlight the importance of identifying GDA events as a mechanism of bacterial resistance to environmental toxins. We demonstrate that amplification events can exist within a population in the absence of selection pressure and without a clear fitness cost. Identifying specific stimuli that trigger these events will help understand factors that accelerate bacterial resistance evolution and have the potential to disrupt the balance of the ecosystem.

microbiology↗

bioRxiv2025

Evaluating the Link Between Efflux Pump Expression and Motility Phenotypes in Pseudomonas aeruginosa Treated with Virulence Inhibitors

Antibiotic resistance continues to rise as a global health threat. Novel anti-virulence strategies diminish the drive for evolutionary pressure, but still hinder a pathogens ability to infect a host. Treatment of the highly virulent Pseudomonas aeruginosa strain PA14 with virulence inhibitors (R-2 and R-6) elicited widely varying transcriptional profiles. Of interest, expression of a family of resistance-nodulation-division (RND) efflux pumps implicated in the intrinsic drug resistance of P. aeruginosa, was significantly altered by R-2 and R-6 treatment. While structurally similar, these inhibitors caused differential expression of various RND efflux pumps within the Mex family--R-2 treatment stimulated expression of mexEF-oprN while R-6 treatment led to increased mexAB-oprM expression. Further expansion into a small library of virulence inhibitors revealed chemical motifs that trigger increases in RND efflux pump expression. Additionally, activation of these efflux pumps suggests low accumulation of virulence inhibitors in WT PA14. Treatment of an efflux pump-deficient strain with R-2 or R-6 resulted in inhibition of several virulence factors, for example R-2 was found to abolish swimming motility. Collectively, treatment with either R-2 or R-6 gives rise to a convoluted transcriptomic response, confounded by the impact of efflux pump expression on the system. However, understanding the moieties that lead to high expression of the efflux pumps enables further rational design of novel virulence inhibitors that do not cause RND efflux pump activation. SummaryAntibiotic resistance continues to rise across the world. The continued failure of antibiotics to treat infectious disease clearly demonstrates the need for new therapeutics. Small molecule inhibitors of virulence, or the ability of a bacteria to establish and maintain an infection, have showed promise in this area. By reducing virulence, the host immune system can clear the infection on its own and there is less selective pressure on the bacteria, reducing the emergence of resistance. Our work demonstrates a clear link between virulence inhibitors and efflux pump expression. Efflux pumps are critical for bacteria to extrude toxic compounds from their interior. Interestingly, the changes in efflux pump expression can be quite large even with similar molecular structures. Additionally, the repression of efflux expression caused by our virulence inhibitors results in synergy with current antibiotics. This work will allow the future design of virulence inhibitors that do not activate efflux pump expression and therefore, enable high intracellular concentrations.

microbiology↗

bioRxiv2024

kinact/KI Value Determination for Penicillin-Binding Proteins in Live Cells

Penicillin-binding proteins (PBPs) are an essential family of bacterial enzymes that are inhibited by the {beta}-lactam class of antibiotics. PBP inhibition disrupts cell wall biosynthesis, which results in deficient growth and proliferation, and ultimately leads to lysis. IC50 values are often employed as descriptors of enzyme inhibition and inhibitor selectivity but can be misleading in the study of time-dependent, irreversible inhibitors. Due to this disconnect, the second order rate constant kinact/KI is a more appropriate metric of covalent inhibitor potency. Despite being the gold standard measurement of potency, kinact/KI values are typically obtained from in vitro assays, which limits assay throughput if investigating an enzyme family with multiple homologs (such as the PBPs). Therefore, we developed a whole-cell kinact/KI assay to define inhibitor potency for the PBPs in Streptococcus pneumoniae using the fluorescent activity-based probe Bocillin-FL. Our results align with in vitro kinact/KI data and show a comparable relationship to previously established IC50 values. These results support the validity of our in vivo kinact/KI method as a means of obtaining a full picture of {beta}-lactam potency for a suite of PBPs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/592586v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@e265ecorg.highwire.dtl.DTLVardef@1917decorg.highwire.dtl.DTLVardef@e2253forg.highwire.dtl.DTLVardef@fb6c60_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Evaluation of Expanded 2-Aminobenzothiazole Library for Inhibition of Pseudomonas aeruginosa Virulence Phenotypes

Bacterial resistance to antibiotics is a rapidly increasing threat to human health. New strategies to combat resistant organisms are desperately needed. One potential avenue is targeting two-component systems, which are the main bacterial signal transduction pathways used to regulate development, metabolism, virulence, and antibiotic resistance. These systems consist of a homodimeric membrane-bound sensor histidine kinase, and a cognate effector, the response regulator. The high sequence conservation in the catalytic and adenosine triphosphate-binding (CA) domain of histidine kinases and their essential role in bacterial signal transduction could enable broad-spectrum antibacterial activity. Through this signal transduction, histidine kinases regulate multiple virulence mechanisms including toxin production, immune evasion, and antibiotic resistance. Targeting virulence, as opposed to development of bactericidal compounds, could reduce evolutionary pressure for acquired resistance. Additionally, compounds targeting the CA domain have the potential to impair multiple two-component systems that regulate virulence in one or more pathogens. We conducted structure-activity relationship studies of 2-aminobenzothiazole-based inhibitors designed to target the CA domain of histidine kinases. We found these compounds have anti-virulence activities in Pseudomonas aeruginosa, reducing motility phenotypes and toxin production associated with the pathogenic functions of this bacterium.

microbiology↗

bioRxiv2023

Penicillin-binding protein redundancy in Bacillus subtilis enables growth during alkaline shock

Penicillin-binding proteins (PBPs) play critical roles in cell wall construction, cell shape, and bacterial replication. Bacteria maintain a diversity of PBPs, indicating that despite their apparent functional redundancy, there is differentiation across the PBP family. Seemingly redundant proteins can be important for enabling an organism to cope with environmental stressors. We sought to evaluate the consequence of environmental pH on PBP enzymatic activity in Bacillus subtilis. Our data show that a subset of B. subtilis PBPs change activity levels during alkaline shock and that one PBP isoform is rapidly modified to generate a smaller protein (i.e., PBP1a to PBP1b). Our results indicate that a subset of the PBPs are preferred for growth under alkaline conditions, while others are readily dispensable. Indeed, we found that this phenomenon could also be observed in Streptococcus pneumoniae, implying that it may be generalizable across additional bacterial species and further emphasizing the evolutionary benefit of maintaining many, seemingly redundant periplasmic enzymes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/533529v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@bf28c7org.highwire.dtl.DTLVardef@e2e88org.highwire.dtl.DTLVardef@1112259org.highwire.dtl.DTLVardef@1e604ae_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Cationic Polymers Enable Internalization of Negatively Charged Chemical Probe into Bacteria

The bacterial cell envelope provides a protective barrier that is challenging for small molecules and biomolecules to cross. Given the anionic nature of both Gram-positive and Gramnegative bacterial cell envelopes, negatively charged molecules are particularly difficult to deliver into these organisms. Many strategies have been employed to penetrate bacteria ranging from reagents such as cell-penetrating peptides, enzymes, and metal-chelating compounds, to physical perturbations. While cationic polymers are known antimicrobial agents, their ability to permeabilize bacterial cells without causing high levels of toxicity and cell lysis has not been demonstrated. Here, we evaluate the ability of four cationic polymers, two linear and two micellar (from self-assembled amphiphilic block copolymers), to facilitate the internalization of an anionic ATP-based chemical probe into Escherichia coli and Bacillus subtilis. Not only did we observe the permeabilization of these organisms, but also found that labeled cells were able to continue to grow and divide. In particular, the micelle-based polymers yielded effective internalization of the negatively charged chemical probe better than their linear analogues.

microbiology↗

bioRxiv2021

The PhoPQ two-component system promotes carbapenem tolerance in Enterobacterales.

Antibiotic tolerance is an understudied potential contributor to antibiotic treatment failure and the emergence of multidrug-resistant bacteria. The molecular mechanisms governing tolerance remain poorly understood. A prominent type of {beta}-lactam tolerance relies on the formation of cell wall-deficient spheroplasts, which maintain structural integrity via their outer membrane (OM), an asymmetric lipid bilayer consisting of phospholipids on the inner leaflet and a lipid-linked polysaccharide (lipopolysaccharide, LPS) enriched in the outer monolayer on the cell surface. How a membrane structure like LPS, with its reliance on mere electrostatic interactions to maintain stability, is capable of countering internal turgor pressure is unknown. Here, we have uncovered a novel role for the PhoPQ two-component system in tolerance to the {beta}-lactam antibiotic meropenem in Enterobacterales. We found that PhoPQ is induced by meropenem treatment and promotes an increase in 4-amino-4-deoxy-L-aminoarabinose [L-Ara4N] modification of lipid A, the membrane anchor of LPS. L-Ara4N modifications enhance structural integrity, and consequently tolerance to meropenem, in several Enterobacterales species. Importantly, mutational inactivation of the negative PhoPQ regulatory element mgrB (commonly selected for during clinical therapy with the last-resort antibiotic colistin, an antimicrobial peptide [AMP]) results in dramatically enhanced tolerance, suggesting that AMPs can collaterally select for meropenem tolerance via stable overactivation of PhoPQ. Lastly, we identify histidine kinase inhibitors (including an FDA-approved drug) that inhibit PhoPQ-dependent LPS modifications and consequently potentiate meropenem to enhance killing of tolerant cells. In summary, our results suggest that PhoPQ-mediated LPS modifications play a significant role in stabilizing the OM, promoting survival when the primary integrity maintenance structure, the cell wall, is removed. SignificanceTreating an infection with an antibiotic often fails, resulting in a tremendous public health burden. One understudied likely reason for treatment failure is the development of "antibiotic tolerance", the ability of bacteria to survive normally lethal exposure to an antibiotic. Here, we describe a molecular mechanism promoting tolerance. A bacterial stress sensor (PhoPQ) is activated in response to antibiotic (meropenem) treatment and consequently strengthens a bacterial protective "shell" to enhance survival. We also identify inhibitors of this mechanism, opening the door to developing compounds that help antibiotics work better against tolerant bacteria.

microbiology↗

bioRxiv2020

Organization of Peptidoglycan Synthesis in Nodes and Separate Rings at Different Stages of Cell Division of Streptococcus pneumoniae

Bacterial peptidoglycan (PG) synthesis requires strict spatial and temporal organization to reproduce specific cell shapes. In the ovoid-shaped, pathogenic bacterium Streptococcus pneumoniae (Spn), septal and peripheral (sidewall-like) PG synthesis occur simultaneously at midcell. To uncover the organization of proteins and activities that carry out these two modes of PG synthesis, we examined Spn cells vertically oriented onto their poles to image the division plane at the high lateral resolution of 3D-SIM (structured-illumination microscopy). Using fluorescent D-amino acid (FDAA) probes, we show that areas of new transpeptidase (TP) activity catalyzed by penicillin-binding proteins (PBPs) separate into a pair of concentric rings early in division, representing peripheral PG (pPG) synthesis (outer ring) and the leading-edge (inner ring) of septal PG (sPG) synthesis. Fluorescently tagged PBP2x or FtsZ locate primarily to the inner FDAA-marked ring, whereas PBP2b and FtsX remain in the outer ring, suggesting roles in sPG or pPG synthesis, respectively. Short pulses of FDAA labeling revealed an arrangement of separate regularly spaced "nodes" of TP activity around the division site of predivisional cells. Control experiments in wild-type and mutant strains support the interpretation of nodal spacing of TP activity, and statistical analysis confirmed that the number of nodes correlates with different ring diameters. This nodal pattern of FDAA labeling is conserved in other ovoid-shaped species. Tagged PBP2x, PBP2b, and FtsX proteins also exhibited nodal patterns with spacing comparable to that of FDAA labeling. Together, these results reveal a highly ordered PG synthesis apparatus in ovococcal bacteria at different stages of division. SIGNIFICANCEThe spatial organization of PBPs and their TP activity at division septa is not well understood. In some bacteria, TP activity and PBP localization seem to be nodal (also called punctate), whereas in other bacteria, discrete foci of PBP activity are infrequently or not observed. Here we report two basic properties of the organization of PBPs and TP activity in the ovoid-shaped bacterium Spn. First, there is distinct spatial separation of the sPG machine, including FtsZ, from the pPG synthesis machine at the midcell of dividing Spn cells. Second, in predivisional cells, PBPs and TP activity are organized heterogeneously into regularly spaced nodes, whose number and dynamic distribution are likely driven by the PG synthesis of PBP:SEDS complexes.

microbiology↗

bioRxiv2019

Harnessing β-Lactam Antibiotics for Illumination of the Activity of Penicillin-Binding Proteins in Bacillus subtilis

Selective chemical probes enable individual investigation of penicillin-binding proteins (PBPs) and provide critical information about their enzymatic activity with spatial and temporal resolution. To identify scaffolds for novel probes to study peptidoglycan biosynthesis in Bacillus subtilis, we evaluated the PBP inhibition profiles of 21 {beta}-lactam antibiotics from different structural subclasses using a fluorescence-based assay. Most compounds readily labeled PBP1, PBP2a, PBP2b or PBP4. Almost all penicillin scaffolds were co-selective for all or combinations of PBP2a, 2b and 4. Cephalosporins, on the other hand, possessed the lowest IC50 values for PBP1 alone or along with PBP4 (ceftriaxone, cefoxitin), 2b (cefotaxime) or 2a, 2b and 4 (cephalothin). Overall, five selective inhibitors for PBP1 (aztreonam, faropenem, piperacillin, cefuroxime and cefsulodin), one selective inhibitor for PBP5 (6-aminopenicillanic acid) and various co-selective inhibitors for other PBPs in B. subtilis were discovered. Surprisingly, carbapenems strongly inhibited PBP3, formerly shown to have low affinity for {beta}-lactams and speculated to be involved in resistance in B. subtilis. To investigate the specific roles of PBP3, we developed activity-based probes based on the meropenem core and utilized them to monitor the activity of PBP3 in living cells. We showed that PBP3 activity localizes as patches in single cells and concentrates as a ring at the septum and the division site during the cell growth cycle. Our activity-based approach enabled spatial resolution of the transpeptidation activity of individual PBPs in this model microorganism, which was not possible with previous chemical and biological approaches.

microbiology↗

bioRxiv2019

Silencing cryptic specialized metabolism in Streptomyces by the nucleoid-associated protein Lsr2

Lsr2 is a nucleoid-associated protein conserved throughout the actinobacteria, including the antibiotic-producing Streptomyces. Streptomyces species encode paralogous Lsr2 proteins (Lsr2 and Lsr2-like, or LsrL), and we show here that of the two, Lsr2 has greater functional significance. We found that Lsr2 binds AT-rich sequences throughout the chromosome, and broadly represses gene expression. Strikingly, specialized metabolic clusters were over-represented amongst its targets, and the cryptic nature of many of these clusters appears to stem from Lsr2-mediated repression. Manipulating Lsr2 activity in model species and uncharacterized isolates resulted in the production of new metabolites not seen in wild type strains. Our results suggest that the transcriptional silencing of biosynthetic clusters by Lsr2 may protect Streptomyces from the inappropriate expression of specialized metabolites, and provide global control over Streptomyces arsenal of signalling and antagonistic compounds.

microbiology↗

bioRxiv2018

Streptomyces volatile compounds influence exploration and microbial community dynamics by altering iron availability

Bacteria and fungi produce a wide array of volatile organic compounds (VOCs), and these can act as infochemicals or as competitive tools. Recent work has shown that the VOC trimethylamine (TMA) can promote a new form of Streptomyces growth, termed exploration. Here, we report that TMA also serves to alter nutrient availability in the area surrounding exploring cultures: TMA dramatically increases the environmental pH, and in doing so, reduces iron availability. This, in turn, compromised the growth of other soil bacteria and fungi. In contrast, Streptomyces thrives in these iron-depleted niches by secreting a suite of differentially modified siderophores, and by upregulating genes associated with siderophore uptake. Further reducing iron levels by siderophore piracy, limiting siderophore uptake, or growing cultures in the presence of iron chelators, unexpectedly enhanced exploration. Our work reveals a new role for VOCs in modulating iron levels in the environment, and implies a critical role for VOCs in modulating the behaviour of microbes and the makeup of their communities.

microbiology↗

bioRxiv2017

PANOPLY: A computational method for identification of promising drugs for a patient based on multidimensional data

PurposeThe majority of cancer patients receive treatments that are minimally informed by omics data. We propose a precision medicine computational framework (PANOPLY: Precision cancer genomic report: single sample inventory) to identify and prioritize drug targets and cancer therapy regimens.\n\nMethodsThe PANOPLY approach integrates clinical data with germline and somatic features obtained from multi-omics platforms, and apply machine learning, and network analysis approaches in the context of the individual patient and matched controls. The PANOPLY workflow employs four steps (i) selection of matched controls to the case of interest (ii) identification of case-specific genomic events (iii) identification of suitable drugs using the driver-gene network and random forest analyses and (iv) provide an integrated multi-omics case report of the patient with prioritization of anti-cancer drugs.\n\nResultsThe PANOPLY workflow can be executed on a stand-alone virtual machine and is also available for download as an R package. We applied the method to an institutional breast cancer neoadjuvant chemotherapy study which collected clinical and genomic data as well as patient-derived xenografts (PDXs) to investigate the prioritization offered by PANOPLY. In a chemotherapy-resistant PDX model, we found that that the prioritized drug, olaparib, was more effective than placebo in treating the tumor (P < 0.05). We also applied PANOPLY to in-house and publicly accessible multi-omics tumor datasets with therapeutic response or survival data available.\n\nConclusionPANOPLY shows promise as a means to prioritize drugs based on clinical and multi-omics data for an individual cancer patient. Additional studies are needed to confirm this approach.

bioinformatics↗