Biology subjects

Flores-Mireles, A. L.

Publications and source records attributed to Flores-Mireles, A. L..

13 recordsLinked to original sources

bioRxiv2026

Liquid-Infused Silicone Catheters Reduce Fungal Burden and Inflammation in Candidozyma auris Bladder Infections.

Candidozyma auris is a high-priority, emerging fungal pathogen frequently isolated from urine in healthcare settings. These isolates are often associated with indwelling urinary catheters, a primary risk factor for catheter-associated urinary tract infections (CAUTIs). Despite its clinical prevalence, the mechanisms of C. auris colonization and pathogenesis within the bladder remain poorly understood. In this study, we screened C. auris isolates from diverse clades using an in vitro biofilm model and in vivo murine models of uncomplicated UTI and CAUTI. While in vitro biofilm formation varied among isolates, the presence of a catheter in vivo significantly enhanced fungal burden in the bladder. Notably, one strain (B11103) caused rapid systemic dissemination and mortality. To address this, we evaluated a liquid-infused silicone (LIS)-catheter coating, which has previously shown efficacy against other uropathogens. The LIS-coating significantly reduced C. auris attachment in vitro and, crucially, mitigated fungal burden on both the catheter and bladder tissue in vivo across all tested strains. For the hyper-virulent B11103 strain, LIS-catheters also significantly reduced dissemination to the kidneys and bloodstream. Furthermore, cytokine analysis revealed that C. auris CAUTI upregulates IL-6, CSF3, and CXCL1; notably, this damaging inflammatory response was dampened by the LIS-catheter. These findings demonstrate that catheterization potentiates C. auris pathogenicity and identify LIS-catheters as a promising, antimicrobial-sparing strategy to prevent colonization, systemic spread, and inflammation during C. auris CAUTI. IMPORTANCEThis research addresses the critical public health challenge posed by the emergence of Candidozyma auris, elucidating its pathogenesis in the urinary tract, the second most common yet understudied reservoir. Here, we find that C. auris exhibits plasticity in its ability to form biofilms in urine and cause uncomplicated UTIs and catheter-associated UTIs. Importantly, we show that our liquid-infused silicone (LIS)-catheters effectively disrupt this cycle by reducing fungal burden, preventing systemic spread, and dampening the damaging host inflammatory response. This work establishes the urinary tract as a critical niche for systemic entry and provides a validated strategy for infection prevention.

microbiology↗

bioRxiv2025

Emergence and spread of outbreak-adapted Candidozyma auris harboring mutations in RBA1

Candidozyma auris is an emerging healthcare-associated fungal pathogen, increasingly isolated from clinical outbreaks, with a high propensity to colonize patients and the medical environment. Here, we leveraged C. auris isolates from multi-facility clinical outbreaks to identify genomic patho-adaptations promoting persistence and dissemination in the healthcare environment. Genomic and phylogenetic analyses revealed loss-of-function mutations in the uncharacterized C. auris transcription factor gene RBA1 to have independently emerged multiple times within these outbreaks. We demonstrate loss of RBA1 increases C. auris adhesion to plastic and human keratinocytes, enhances biofilm formation, and exacerbates fungal burden in a mouse model of catheter-associated urinary tract infection. Finally, we uncover mutations in RBA1 have emerged during multiple C. auris outbreaks across the globe, and that RBA1 mutant lineages are present among large ongoing clinical outbreaks. These results reveal loss-of-function mutations in C. auris RBA1 as a novel and clinically relevant genetic determinant of enhanced outbreak characteristics.

microbiology↗

bioRxiv2025

Characterization of a Novel Cell Wall-Associated Nucleotidase of Enterococcus faecalis that Degrades Extracellular c-di-AMP

Enterococcus faecalis is a prolific opportunistic pathogen responsible for a range of life-threatening infections for which treatment options are increasingly limited due to the high prevalence of multidrug-resistant isolates. Cyclic di-AMP has emerged as an essential bacterial signaling molecule due to its impact on physiological processes, including osmotic adaptation, cell wall homeostasis, antibiotic tolerance, and virulence. In addition, c-di-AMP is a potent pathogen-associated molecular pattern (PAMP) molecule recognized by the host immune system to trigger protective responses. In previous work, we identified and characterized the enzymes responsible for the synthesis and degradation of intracellular c-di-AMP in E. faecalis, demonstrating that maintaining c-di-AMP homeostasis is vital for bacterial fitness and virulence. In addition to the intracellular enzymes that regulate c-di-AMP levels, a limited number of bacteria encode surface-associated nucleotidases capable of cleaving extracellular c-di-AMP, potentially facilitating immune evasion. Here, we characterize a novel and unique cell wall-anchored phosphodiesterase, termed EecP (E. faecalis extracellular c-di-AMP phosphodiesterase), which features duplicated catalytic domains and specifically degrades extracellular c-di-AMP. Deletion of eecP ({Delta}eecP) resulted in a marked accumulation of extracellular c-di-AMP. Although the {Delta}eecP strain exhibited comparable growth and behavior to the parent strain in vitro, it displayed increased susceptibility to killing by phagocytic cells. Using two murine infection models, we show that the impact of eecP deletion and the consequent buildup of extracellular c-di-AMP on E. faecalis pathogenesis may be site-specific. Notably, disseminated infection was more severe in mice infected with the {Delta}eecP strain, suggesting that extracellular c-di-AMP influences infection outcomes, likely through modulation of host immune responses. Author SummaryEnterococcus faecalis is a major opportunistic pathogen and a leading cause of several life-threatening hospital-associated infections. Cyclic di-AMP is a bacterial second messenger nucleotide that regulates essential cellular processes and plays key roles in bacterial pathogenesis and host immune activation. We previously characterized the enzymes responsible for the synthesis and degradation of c-di-AMP in E. faecalis, demonstrating that this signaling molecule is crucial for bacterial fitness and virulence. In this study, we describe the characterization of EecP, a novel cell wall-associated enzyme that degrades c-di-AMP extracellularly. Our findings identify EecP as a new virulence factor in E. faecalis, capable of modulating infection outcomes.

microbiology↗

bioRxiv2025

Astrocyte and neurogenic mechanisms of protective gene therapy prevent brain disease and death due to non-ketotic hyperglycinemia

Genetic defects in glycine decarboxylase (GLDC) cause non-ketotic hyperglycinemia (NKH), a rare and frequently fatal neurometabolic disease, which lacks FDA-approved therapies. We characterized CRISPR Cas9-edited humanized mice expressing a prevalent clinical mutation after administration with a single intraperitoneal dose of a novel recombinant of adeno-associated viral vector 9 expressing GLDC (rAAV9-GLDC). Long term biological activity of rAAV9-GLDC was first validated by assessment of its systemic efficacy over five and ten months in mice. Access of rAAV9 to the brain was confirmed by tracking green fluorescent protein (GFP) after a single intraperitoneal dose of rAAV9-GFP. Over five months, control mock treated GFP-mice showed reduction in astrocytes but not microglia, oligodendrocytes or neurons in the brain. 37% of these animals suffered long term neurological disease and/or death. rAAV9-GLDC boosts astrogenesis without triggering an inflammatory response and confers 100% protection against disease progression and fatality due to NKH.

neuroscience↗

bioRxiv2024

The catheterized bladder environment induces dysregulation of macrophage polarization exacerbating bacterial UTI

Urinary catheterization causes bladder damage, predisposing hosts to catheter-associated urinary tract infections (CAUTIs). CAUTI pathogenesis is mediated by bladder damage-induced inflammation, resulting in accumulation and deposition of the blood-clotting protein fibrinogen (Fg) and its matrix form fibrin, which are exploited by uropathogens as biofilm platforms to establish infection. Catheter-induced inflammation also results in robust immune cell recruitment, including macrophages (M{phi}s). A fundamental knowledge gap is understanding the mechanisms by which the catheterized-bladder environment suppresses the M{phi} antimicrobial response, allowing uropathogen persistence. Here, we found that Fg and fibrin differentially modulate M1 and M2 M{phi} polarization, respectively. We unveiled that fibrin accumulation in catheterized mice induced an anti-inflammatory M2-like M{phi} phenotype, correlating with pathogen persistence. Even GM-CSF treatment of wildtype mice to promote M1 polarization was not sufficient to reduce bacterial burden and dissemination, indicating that the catheterized-bladder environment provides mixed signals, dysregulating M{phi} polarization, hindering its antimicrobial response against uropathogens.

immunology↗

bioRxiv2023

Group A Streptococcus remains viable inside fibrin clots and gains access to human plasminogen for subsequent fibrinolysis and dissemination

Group A Streptococcus (GAS) is a Gram-positive bacterial pathogen that causes a wide spectrum of illnesses ranging from pharyngitis and rheumatic fever to more invasive and severe diseases such as necrotizing fasciitis and toxic shock syndrome. Invasive outcomes of GAS infections often result from entry of the bacteria via an open wound into tissue and blood systems. The coagulation cascade serves as an innate defense mechanism that initiates fibrin clots to sequester bacteria and restrict its growth and prevent dissemination into deeper tissues. GAS, especially skin-tropic bacterial strains, utilize the specific virulence factors Plasminogen binding M-protein (PAM) and streptokinase (SK) to manipulate hemostasis and ultimately activate human plasminogen to cause fibrinolysis and escape from the fibrin clot. A major unresolved question regarding this process is to understand the temporal dynamics of how GAS that is enmeshed in a fibrin clot accesses host plasminogen for dissolution of the clot and eventual dissemination. Using fluorescently labeled plasminogen and fibrinogen, we established conditions to observe the process of fibrin clot dissolution by GAS (an AP53 CovR+S-strain) that is sequestered in a fibrin clot using real-time imaging microscopy. We hypothesized that initiation of fibrinolysis by GAS inside a fibrin clot would be determined by the rate of hPg access into the fibrin clot where bacteria are trapped. Our live imaging studies show that GAS trapped inside a fibrin clot, has limited access to hPg; however, at 4.25 h post incubation, when sufficient hPg is accessible to the bacterium, fibrinolysis quickly occurs. If hPg is bound to the bacterial surface prior to being trapped inside a clot, dissolution and bacterial dissemination occurs at a much faster rate of 2.5 h post incubation. During the time which bacteria are trapped in the clot without access to hPg, we did not observe any growth of GAS; however, we demonstrate that the bacteria continue to remain viable inside the fibrin clot. We performed RNA-seq analysis of GAS and the isogenic GAS SK-deficient mutant to understand SK-dependent transcriptional changes during the lag-phase of the GAS bacteria inside the fibrin clot. We observed a dramatic change in the transcription profile of wt GAS inside the fibrin clot over time prior to escape from the fibrin clot (22 gene expression changes at 4h, to 802 gene expression changes at 8h). Furthermore, we also identified gene expression changes that were distinct between wt GAS and the GAS SK-deficient mutant. Our findings reveal for the first time that GAS can engage a latent, growth suspended phase whereby physical structures such as fibrin clots and Neutrophil extracellular traps that immobilize an invading pathogen allow bacteria to remain viable and transcriptionally active for an extended time during host infection. GAS that is trapped in a fibrin clot will therefore enter a state in which the bacteria suspend growth, but remain viable, until sufficient access to hPg allow it to initiate fibrinolysis and escape into surrounding tissues. The viability of GAS while trapped and its readiness to avoid immune defenses allow GAS to act quickly to disseminate when host conditions are more favorable for the bacteria.

microbiology↗

bioRxiv2022

Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans

For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans, which employs a guide RNA to target an activator complex to the promoter region of a gene of interest, thus driving transcriptional expression of that gene. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust and constitutive overexpression. We further demonstrate the specificity of the system via RNA sequencing. We highlight the application of CRISPRa to overexpress genes involved in pathogenesis and drug resistance and contribute towards the identification of novel phenotypes. Together, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.

microbiology↗

bioRxiv2021

Inhibiting Host Protein Deposition on Urinary Catheters Reduces Urinary Tract Infections

Microbial adhesion to medical devices is common for hospital-acquired infections, particularly for urinary catheters. If not properly treated these infections cause complications and exacerbate antimicrobial resistance. Catheter use elicits bladder inflammation, releasing host serum-proteins, including fibrinogen, into the bladder, which deposit on the urinary catheter. Enterococcus faecalis uses fibrinogen as a scaffold to bind and persist in the bladder despite antibiotic treatments. Inhibition of fibrinogen-pathogen interaction significantly reduces infection. Here, we show deposited fibrinogen is advantageous for uropathogens, suggesting that targeting catheter protein deposition may reduce colonization creating an effective intervention. Hostprotein deposition was reduced, using liquid-infused catheters, resulting in decreased colonization on catheters, in bladders, and dissemination in vivo. Furthermore, proteomics revealed a significant decrease in deposition of host-secreted proteins on liquid-infused catheter surfaces. Our findings suggest targeting microbial binding scaffolds may be an effective antibiotic-sparing intervention for use against catheter-associated urinary tract infections and other medical device infections.

microbiology↗

bioRxiv2021

The AdcACB/AdcAII system is Essential for Zinc Homeostasis and an Important Contributor of Enterococcus faecalis Virulence

Bacterial pathogens require a variety of micronutrients for growth, including trace metals such as iron, manganese, and zinc (Zn). Despite their relative abundance in host environments, access to these metals is severely restricted during infection due to host-mediated defense mechanisms collectively known as nutritional immunity. Despite a growing appreciation of the importance of Zn in host-pathogen interactions, the mechanisms of Zn homeostasis and the significance of Zn to the pathophysiology of E. faecalis, a major pathogen of nosocomial and community-associated infections, have not been investigated. Here, we show that E. faecalis encoded an ABC-type transporter AdcACB and an orphan substrate-binding lipoprotein AdcAII that work cooperatively to maintain Zn homeostasis. Simultaneous inactivation of adcA and adcAII or the entire adcACB operon led to significant reduction in intracellular Zn under Zn-restricted conditions, heightened sensitivity to Zn-chelating agents including human calprotectin, aberrant cell morphology, and impaired fitness in serum ex vivo. Additionally, inactivation of adcACB and adcAII significantly reduced bacterial tolerance towards cell envelope-targeting antibiotics, which may be associated to altered fatty acid abundance and species. Lastly, we show that the AdcACB/AdcAII system contributes to E. faecalis virulence in an invertebrate (Galleria mellonella) infection model and in two catheter-associated mouse infection models that recapitulate many of the host conditions associated with enterococcal human infections. Collectively, this report reveals that high-affinity Zn import is essential for the pathogenesis of E. faecalis indicating that the surface-associated AdcA and AdcAII lipoproteins are potential therapeutic targets.

microbiology↗

bioRxiv2021

Esrrγa regulates nephron development and ciliogenesis by controlling prostaglandin synthesis and cooperation with Ppargc1a

Cilia are essential for the ontogeny and function of many tissues, including the kidney. In mammals, Esrr{gamma} has been previously established as a significant determinant of renal health, with decreased expression linked to age related dysfunction, cyst formation, and kidney disease. Here, we report that the Esrr{gamma} vertebrate ortholog estrogen related receptor gamma a (esrr{gamma}a) is essential for proper cell fate choice within kidney functional units (nephrons) as well as ciliogenesis. Deficiency of esrr{gamma}a resulted in nephrons with alterations in proximodistal segmentation and a decreased multiciliated epithelial cell populace. Surprisingly, esrr{gamma}a deficiency disrupted renal ciliogenesis and caused a similar abrogation within the developing node and otic vesicle--all defects that occurred independently of changes in cell polarity or basal body organization. These phenotypes were consistent with interruptions in prostaglandin signaling, and we found that ciliogenesis was rescued in esrr{gamma}a deficient embryos with exogenous PGE2 or through overexpression of the cyclooxygenase enzyme Ptgs1. Through genetic interaction studies, we found that peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (ppargc1a), which acts upstream of Ptgs1-mediated prostaglandin synthesis, has a synergistic relationship with esrr{gamma}a in the ciliogenic pathway. These data position esrr{gamma}a as a novel link between ciliogenesis and nephrogenesis through regulation of prostaglandin signaling and cooperation with ppargc1a, and highlight esrr{gamma}a as a potential new target for future ciliopathic treatments.

developmental biology↗

bioRxiv2021

Catheterized-bladder environment induces hyphal Candida albicans formation, promoting fungal colonization and persistence.

Catheter-associated urinary tract infections (CAUTIs) account for 40% of all hospital-acquired infections. Given that 20-50% of all hospitalized patients receive a catheter, CAUTIs are one of the most common hospital-acquired infections and a significant medical complication as they result in increased morbidity, mortality, and an estimated annual cost of $340-370 million. Candida spp. - specifically Candida albicans - are a major causative agent of CAUTIs (17.8%), making it the second most common CAUTI uropathogen. Despite this frequent occurrence, the cellular and molecular details of C. albicans infection in the CAUTI microenvironment are poorly understood. Here, we characterize fungal virulence mechanisms and fungal biofilm formation during CAUTI for the first time. We found that the catheterized bladder environment triggers Candida virulence programs and robust biofilm formation through Efg1-dependent hyphal morphogenesis and Als1, an Efg1-downstream effector. Additionally, we show that the adhesin Als1 is necessary for in vitro and in vivo C. albicans biofilm formation dependent on the presence of fibrinogen (Fg), a coagulation factor released in the bladder due to the mechanical damage caused by urinary catheterization. Furthermore, in the presence of Fg, overexpression of ALS1 in C. albicans led to enhanced colonization and dissemination, while deletion of ALS1 reduced both outcomes during CAUTIs. Our study ultimately unveils the mechanism that contributes to fungal CAUTI, which may provide more effective targets for future therapies to prevent these infections.

microbiology↗

bioRxiv2019

(p)ppGpp and CodY promote Enterococcus faecalis virulence in a murine model of catheter-associated urinary tract infection

In Firmicutes, the nutrient-sensing regulators (p)ppGpp, the effector molecule of the stringent response, and CodY work in tandem to maintain bacterial fitness during infection. Here, we tested (p)ppGpp and codY mutant strains of Enterococcus faecalis in a catheter-associated urinary tract infections (CAUTI) mouse model and used global transcriptional analysis to investigate the (p)ppGpp and CodY relationship. Absence of (p)ppGpp or single inactivation of codY led to lower bacterial loads in catheterized bladders, and diminished biofilm formation on fibrinogen-coated surfaces under in vitro and in vivo conditions. Single inactivation of the bifunctional (p)ppGpp synthetase/hydrolase rel did not affect virulence supporting previous evidence that association of (p)ppGpp with enterococcal virulence is not dependent on activation of the stringent response. Inactivation of codY in the (p)ppGpp0 strain restored E. faecalis virulence in the CAUTI model as well as the ability to form biofilms in vitro. Transcriptome analysis revealed that inactivation of codY restores, for the most part, the dysregulated metabolism of (p)ppGpp0 cells. While a clear linkage between (p)ppGpp and CodY with expression of virulence factors could not be established, targeted transcriptional analysis indicate that a possible association between (p)ppGpp and c-di-AMP signaling pathways in response to the conditions found in the bladder may plays a role in enterococcal CAUTI. Collectively, this study identifies the (p)ppGpp-CodY network as an important contributor to enterococcal virulence in catheterized mouse bladder and supports that basal (p)ppGpp pools and CodY promote virulence through maintenance of a balanced metabolism during adverse conditions.\n\nImportanceCatheter-associated urinary tract infections (CAUTI) are one of the most frequent types of infection found in the hospital setting that can develop into serious and potentially fatal bloodstream infections. One of the infectious agents that frequently cause complicated CAUTI is the bacterium Enterococcus faecalis, a leading cause of hospital-acquired infections that are often difficult to treat due to the exceptional multidrug resistance of some isolates. Understanding the mechanisms by which E. faecalis causes CAUTI will aid in the discovery of new druggable targets to treat these infections. In this study, we report the importance of two nutrient-sensing bacterial regulators, named (p)ppGpp and CodY, for the ability of E. faecalis to infect the catheterized bladder of mice.

microbiology↗

bioRxiv2018

Manganese acquisition is essential for virulence of Enterococcus faecalis

Manganese (Mn) is an essential micronutrient that is not readily available to pathogens during infection due to an active host defense mechanism known as nutritional immunity. To overcome this nutrient restriction, bacteria utilize high-affinity transporters that allow them to compete with host metal-binding proteins. Despite the established role of Mn in bacterial pathogenesis, little is known about the relevance of Mn in the pathophysiology of E. faecalis. Here, we identified and characterized the major Mn acquisition systems of E. faecalis. We discovered that the ABC-type permease EfaCBA and two Nramp-type transporters, named MntH1 and MntH2, work collectively to promote cell growth under Mn-restricted conditions. The simultaneous inactivation of EfaCBA, MntH1 and MntH2 ({Delta}efa{Delta}mntH1{Delta}mntH2 strain) led to drastic reductions (>95%) in intracellular Mn content, severe growth defects in body fluids (serum and urine) ex vivo, significant loss of virulence in Galleria mellonella, and virtually complete loss of virulence in rabbit endocarditis and murine catheter-associated urinary tract infection (CAUTI) models. Despite the functional redundancy of EfaCBA, MntH1 and MntH2 under in vitro or ex vivo conditions and in the invertebrate model, dual inactivation of efaCBA and mntH2 ({Delta}efa{Delta}mntH2 strain) was sufficient to prompt maximal sensitivity to calprotectin, a Mn- and Zn-chelating host antimicrobial protein, and for the loss of virulence in mammalian models. Interestingly, EfaCBA appears to play a prominent role during systemic infection, whereas MntH2 was more important during CAUTI. The different roles of EfaCBA and MntH2 in these sites could be attributed, at least in part, to the differential expression of efaA and mntH2 in cells isolated from hearts or from bladders. Collectively, this study demonstrates that Mn acquisition is essential for the pathogenesis of E. faecalis and validates Mn uptake systems as promising targets for the development of new antimicrobials.

microbiology↗