Biology subjects

Richardson, A. R.

Publications and source records attributed to Richardson, A. R..

9 recordsLinked to original sources

bioRxiv2026

ST6Gal2 promotes α2,6-sialylation and aggressive phenotypes in neuroblastoma cells

Neuroblastoma is the most common extracranial solid tumor of childhood. Its clinical behavior ranges from spontaneous regression to lethal, treatment-refractory disease. Aberrant 2,6-sialylation contributes to aggressive phenotypes in many cancers, but the role of ST6Gal2, a neural-enriched 2,6-sialyltransferase, in neuroblastoma is largely unexplored. Here, we examine the clinical and functional significance of ST6Gal2 in neuroblastoma. In two independent public cohorts (SEQC, n=498; Kocak, n=649), high ST6GAL2 expression was associated with significantly worse overall and event-free survival. In the SEQC cohort, ST6GAL2 expression was higher in high-risk and MYCN-amplified tumors, varied across International Neuroblastoma Staging System stages, and correlated positively with a mesenchymal transcriptional signature (Spearman {rho}=0.181). The mesenchymal correlation was reproduced in the Kocak cohort ({rho}=0.204). Stable shRNA-mediated knockdown of ST6GAL2 in SK-N-AS and SK-N-BE(2) cells reduced proliferation and viability, impaired wound closure, and decreased migration and invasion. In preliminary experiments in SK-N-AS cells, ST6GAL2 knockdown reduced binding of Sambucus nigra agglutinin, consistent with a role for ST6Gal2 in 2,6-sialylation. Together, these findings link ST6Gal2 expression to aggressive clinical and transcriptional features and pro-tumorigenic phenotypes in neuroblastoma and nominate ST6Gal2-mediated sialylation as a candidate pathway for mechanistic study.

cancer biology↗

bioRxiv2025

Methicillin-Resistant Staphylococcus aureus has Phenotypic Variation in mecA Expression that Alters Antibiotic Sensitivity

Methicillin resistant Staphylococcus aureus (MRSA) sepsis has a high rate of morbidity and mortality. Multiple clinical studies have demonstrated improved outcomes when MRSA sepsis is treated with dual antibiotic therapy that includes a {beta}-lactam antibiotic such as cefazolin. This is a paradox as MRSA should be inherently resistant to this class of antibiotics. We report a serendipitous observation revealed a phenotype where MRSA became sensitive to cefazolin when cultured in a physiologic relevant media of fetal bovine serum as well as in synovial fluid. This could be observed across multiple clinical isolates. Expected resistance was maintained when cultured in Muller Hinton Broth (MHB). MRSA {beta}-lactam antibiotic resistance is mediated by PBP2a, a penicillin-binding protein encoded by mecA. We hypothesized that this phenotype of antibiotic sensitivity in physiologic medium was based, in part, on levels of PBP2a expression and post-translational modifications of peptidoglycan wall teichoic acid (WTA). We therefore conducted quantitative RT-PCR analysis and Western blotting which demonstrated limited mecA expression in the mRNA level and limited PBP2a protein level when cultured in FBS or synovial fluid as compared to the clinical microbiology standard MHB, respectively. Whole genome sequencing of loss of function mutants generated through serial passaging in FBS revealed that the clp family of proteins and rpo genes were involved in {beta}-lactam resistance. Cell wall peptidoglycan analysis suggested that WTA glycosylation was altered between {beta}-lactam resistant and sensitive MRSA phenotypes. Together, this suggests pathways for clpP, rpoB, and WTA glycosylation can be new potential targets for MRSA treatment. IMPORTANCEThis study demonstrates that MRSA has phenotypic variation in mecA expression, PBP2a protein levels, and patterns of wall teichoic acid glycosylation based on environmental conditions which can result in a phenotype of {beta}-lactam antibiotic sensitivity. The presence of an antibiotic resistance gene does not necessarily result in antibiotic resistance. This provides a possible explanation for the clinical observations of potential therapeutic benefit of {beta}-lactam antibiotics in dual therapy treatment of MRSA sepsis and suggests new therapeutic strategies to combat multidrug resistant bacteria. More importantly, identifying genes potentially responsible for this phenotype offers new potential therapeutic targets for the treatment of multidrug resistant infection.

microbiology↗

bioRxiv2024

Microbiota and metabolic adaptation shape Staphylococcus aureus virulence and antimicrobial resistance during intestinal colonization

Depletion of microbiota increases susceptibility to gastrointestinal colonization and subsequent infection by opportunistic pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). How the absence of gut microbiota impacts the evolution of MRSA is unknown. The present report used germ-free mice to investigate the evolutionary dynamics of MRSA in the absence of gut microbiota. Through genomic analyses and competition assays, we found that MRSA adapts to the microbiota-free gut through sequential genetic mutations and structural changes that enhance fitness. Initially, these adaptations increase carbohydrate transport; subsequently, evolutionary pathways largely diverge to enhance either arginine metabolism or cell wall biosynthesis. Increased fitness in arginine pathway mutants depended on arginine catabolic genes, especially nos and arcC, which promote microaerobic respiration and ATP generation, respectively. Thus, arginine adaptation likely improves redox balance and energy production in the oxygen-limited gut environment. Findings were supported by human gut metagenomic analyses, which suggest the influence of arginine metabolism on colonization. Surprisingly, these adaptive genetic changes often reduced MRSAs antimicrobial resistance and virulence. Furthermore, resistance mutation, typically associated with decreased virulence, also reduced colonization fitness, indicating evolutionary trade-offs among these traits. The presence of normal microbiota inhibited these adaptations, preserving MRSAs wild-type characteristics that effectively balance virulence, resistance, and colonization fitness. The results highlight the protective role of gut microbiota in preserving a balance of key MRSA traits for long-term ecological success in commensal populations, underscoring the potential consequences on MRSAs survival and fitness during and after host hospitalization and antimicrobial treatment. ImportanceThe fitness of MRSA depends on its ability to colonize. A key, underappreciated observation is that gut colonization frequently serves as the site for MRSA infections, especially among vulnerable groups such as children and hospitalized adults. By evolving MRSA strains in germ-free mice, we identify molecular mechanisms underlying how MRSA exploits a depletion in host microbiota to enhance gut colonization fitness. This work points to bacterial colonization factors that may be targetable. Our findings indicate that adaptive changes in MRSA often reduce its antimicrobial resistance and virulence, and are suppressed by the presence of native commensal bacteria. This work helps explain the ecology of pathoadaptive variants that thrive in hospital settings but falter under colonization conditions in healthy hosts. Additionally, it illustrates the potential adverse effects of prolonged, broad-spectrum empirical antimicrobial therapy and adds a new type of weight to calls for microbiota transplantation to reduce colonization by antimicrobial-resistant pathogens.

microbiology↗

bioRxiv2023

Quorum-sensing agr system of Staphylococcus aureus primes gene expression for protection from lethal oxidative stress

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H2O2, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr increased both respiration and fermentation but decreased ATP levels and growth, suggesting that {Delta}agr cells assume a hyperactive metabolic state in response to reduced metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of {Delta}agr strains to lethal H2O2 doses. Increased survival of wild-type agr cells during H2O2 exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected {Delta}agr cells from killing by H2O2. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived "memory" of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Nox2-/-) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage.

microbiology↗

bioRxiv2022

Adaptation to overflow metabolism by mutations that impair tRNA modification in experimentally evolved bacteria

When microbes grow in foreign nutritional environments, selection may enrich mutations in unexpected pathways connecting growth and homeostasis. An evolution experiment designed to identify beneficial mutations in Burkholderia cenocepacia captured six independent nonsynonymous substitutions in the essential gene tilS, which modifies tRNAIle2 by adding a lysine to the anticodon for faithful AUA recognition. Further, five additional mutants acquired mutations in tRNAIle2, which strongly suggests that disrupting the TilS:tRNAIle2 interaction was subject to strong positive selection. Mutated TilS incurred greatly reduced enzymatic function but retained capacity for tRNAIle2 binding. Yet both mutant sets outcompeted wild-type by decreasing lag phase duration by [~]3.5 hours. We hypothesized that lysine demand could underlie fitness in the experimental conditions. As predicted, supplemental lysine complemented the ancestral fitness deficit, but so did additions of several other amino acids. Mutant fitness advantages were also specific to rapid growth on galactose using oxidative overflow metabolism that generates redox imbalance, not resources favoring more balanced metabolism. Remarkably, 13 tilS mutations also evolved in the Long-Term Evolution Experiment with E. coli, including four fixed mutations. These results suggest that TilS or unknown binding partners contribute to improved growth under conditions of rapid sugar oxidation at the predicted expense of translational accuracy.

microbiology↗

bioRxiv2022

Novel Requirement for Staphylococcal Cell Wall-Anchored Protein SasD in Pulmonary Infection

Staphylococcus aureus can complicate preceding viral infections, including influenza virus. A bacterial infection combined with a preceeding viral infection, known as super-infection, leads to worse outcomes compared to single infection. Most of the super-infection literature focuses on the changes in immune responses to bacteria between homeostatic and virally infected lungs. However, it is unclear how much of an influence bacterial virulence factors have in super-infection. Staphylococcal species express a broad range of cell wall-anchored proteins (CWAs) that have roles in host adhesion, nutrient acquisition, and immune evasion. We screened the importance of these CWAs using mutants lacking individual CWAs in vivo in both bacterial pneumonia and influenza super-infection. In bacterial pneumonia, lacking individual CWAs led to varying decreases in bacterial burden, lung damage, and immune infiltration into the lung. However, the presence of a preceding influenza infection partially abrogated the requirement for CWAs. In the screen, we found that the uncharacterized CWA S. aureus surface protein D (SasD) induced changes in both inflammatory and homeostatic lung markers. We further characterized a SasD mutant (sasD A50.1) in the context of pneumonia. Mice infected with sasD A50.1 had decreased bacterial burden, inflammatory responses, and mortalty compared to wildtype S. aureus. Mice also had reduced levels of IL-1{beta} compared with wildtype, likely derived from macrophages. Reductions in IL-1{beta} transcript levels as well as increased macrophage viability implicate altered macrophage cell death pathways. These data identify a novel virulence factor for S. aureus that influences inflammatory signaling within the lung. ImportanceStaphylococcus aureus is a common commensal bacteria that can cause severe infections, such as pneumonia. In the lung, viral infections increase the risk of staphylococcal pneumonia, leading to combined infections known as super-infections. The most common virus associated with S. aureus pneumonia is influenza, and super-infections lead to worse patient outcomes compared to either infection alone. While there is much known about how the immune system differs between healthy and virally infected lungs, the role of bacterial virulence factors in super-infection is less understood. The significance of our research is identifying new bacterial virulence factors that play a role in the initiation of infection and lung injury, which could lead to future therapies to prevent pulmonary single or super-infection with S. aureus.

microbiology↗

bioRxiv2021

The nutritional environment is sufficient to select coexisting biofilm and quorum-sensing mutants of Pseudomonas aeruginosa

The evolution of bacterial populations during infections can be influenced by various factors including available nutrients, the immune system, and competing microbes, rendering it difficult to identify the specific forces that select on evolved traits. The genomes of Pseudomonas aeruginosa isolated from the airway of patients with cystic fibrosis (CF), for example, have revealed commonly mutated genes, but which phenotypes led to their prevalence is often uncertain. Here, we focus on effects of nutritional components of the CF airway on genetic adaptations by P. aeruginosa grown in either well-mixed (planktonic) or biofilm-associated conditions. After only 80 generations of experimental evolution in a simple medium with glucose, lactate, and amino acids, all planktonic populations diversified into lineages with mutated genes common to CF infections: morA, encoding a regulator of biofilm formation, or lasR, encoding a quorum sensing regulator that modulates the expression of virulence factors. Although mutated quorum sensing is often thought to be selected in vivo due to altered virulence phenotypes or social cheating, isolates with lasR mutations demonstrated increased fitness when grown alone and outcompeted the ancestral PA14 strain. Nonsynonymous SNPs in morA increased fitness in a nutrient concentration-dependent manner during planktonic growth and surprisingly also increased biofilm production. Populations propagated in biofilm conditions also acquired mutations in loci associated with chronic infections, including lasR and cyclic-di-GMP regulators roeA and wspF. These findings demonstrate that nutrient conditions and biofilm selection are alone sufficient to select mutants with problematic clinical phenotypes including increased biofilm and altered quorum sensing. ImportancePseudomonas aeruginosa produces dangerous chronic infections that are known for their rapid diversification and recalcitrance to treatment. We performed evolution experiments to identify adaptations selected by two specific aspects of the CF respiratory environment: nutrient levels and surface attachment. Propagation of P. aeruginosa in nutrients present within the CF airway was alone sufficient to drive diversification into subpopulations with identical mutations in regulators of biofilm and quorum sensing to those arising during infection. Thus, the adaptation of opportunistic pathogens to nutrients found in the host may select mutants with phenotypes that complicate treatment and clearance of infection.

microbiology↗

bioRxiv2020

Genotypic diversity between surgical and nasal Staphylococcus aureus isolates

Staphylococcus aureus is a common organism in periprosthetic joint infection (PJI). Little is known about S. aureus genetic diversity in PJI as compared to nasal carriage. We hypothesized PJI S. aureus strains would be associated with increased virulence as compared to those from nasal carriage. Whole genome sequencing and multilocus sequence typing (MLST) was performed to genotype these two populations at high resolution. MLST revealed a variety of genotypes in both populations but many belonged to the most common clonal complexes. In nasal cultures, 69% of strains were of clonal complexes CC5, CC8, and CC30. In PJI cultures, only 51% could be classified in these common clonal complexes. Remaining strains were atypical, and these atypical strains in PJI were associated with poor host status and compromised immune conditions. Mutations in genes involved in fibronectin binding (ebh, fnbA, clfA, clfB) systematically distinguished later PJI isolates from the first PJI isolate from each patient. S. aureus isolated from nasal carriage and PJI specimens differ significantly, with the latter being more diverse. Strains associated with lower pathogenicity tended to be found in immunocompromised patients, suggesting the host immune system plays an important role in preventing PJI. Repeated mutations in S. aureus genes associated with extracellular matrix binding were identified suggesting an adaptive, parallel evolution in S. aureus during the development of PJI.

microbiology↗

bioRxiv2019

The Toxin Antitoxin MazEF Drives Staphylococcus aureus Chronic Infection

Staphylococcus aureus is the major organism responsible for surgical implant infections. Antimicrobial treatment of these infections often fails leading to expensive surgical intervention and increased risk of mortality to the patient. The challenge in treating these infections is associated with the high tolerance of S. aureus biofilm to antibiotics. MazEF, a toxin-antitoxin system, is thought to be an important regulator of this phenotype, but its physiological function in S. aureus is controversial. Here, we examined the role of MazEF in developing chronic infections by comparing growth and antibiotic tolerance phenotypes in three S. aureus strains to their corresponding strains with disruption of mazF expression. Strains lacking mazF production showed increased biofilm growth, and decreased biofilm antibiotic tolerance. Deletion of icaADBC in the mazF::tn background suppressed the growth phenotype observed with mazF-disrupted strains, suggesting the phenotype was ica-dependent. We confirmed these phenotypes in our murine animal model. Loss of mazF resulted in increased bacterial burden and decreased survival rate compared to its wild-type strain demonstrating that loss of the mazF gene caused an increase in S. aureus virulence. Although lack of mazF gene expression increased S. aureus virulence, it was more susceptible to antibiotics in vivo. Combined, the ability of mazF to inhibit biofilm formation and promote biofilm antibiotic tolerance plays a critical role in transitioning from an acute to chronic infection that is difficult to eradicate with antibiotics alone.\n\nImportanceSurgical infections are one of the most common types of infections obtained in a hospital. Staphylococcus aureus is the most common pathogen associated with this infection. These infections are resilient and difficult to eradicate as the bacteria form a biofilm, a community of bacteria held together by an extracellular matrix. Compared to bacteria floating in liquid, bacteria in a biofilm are more resistant to antibiotics. The mechanism behind how bacteria develop this resistance and establish a chronic infection is unknown. We demonstrate that mazEF, a toxin-antitoxin gene, inhibits biofilm formation and promotes biofilm antibiotic tolerance which allows S. aureus to transition from an acute to chronic infection that cannot be eradicated with antibiotics but is less virulent. This gene not only makes the bacteria more tolerant to antibiotics but makes the bacteria more tolerant to the host.

microbiology↗