Biology subjects

Pimentel, C.

Publications and source records attributed to Pimentel, C..

7 recordsLinked to original sources

bioRxiv2026

A Nickel N-Heterocyclic Biscarbene Complex Derived from Caffeine Enhances Fluconazole Efficacy against Candida glabrata

Invasive infections caused by Candida spp. are associated with high morbidity and mortality, particularly in immunocompromised patients, and are increasingly difficult to treat due to rising antifungal resistance. Here, we further investigate the antifungal properties of a previously reported nickel N-heterocyclic biscarbene derived from caffeine (compound 5) and describe the synthesis of an analogous nickel N-heterocyclic biscarbene based on benzimidazole (compound 8), designed to evaluate the impact of the biscarbene and xanthine frameworks on activity and toxicity. Compound 5 displayed selective activity against Candida glabrata, inhibited biofilm formation and showed greater cellular accumulation in this species. In a Galleria mellonella infection model, 5 significantly reduced fungal burden while exhibiting lower cytotoxicity than the benzimidazole analogue 8. Importantly, although compound 5 and fluconazole are individually fungistatic, their combination was fungicidal against C. glabrata. In the presence of compound 5, the minimal inhibitory concentration of fluconazole decreased against both a fluconazole-resistant petite mutant and a respiratory-competent C. glabrata strain evolved in vitro to fluconazole resistance. Compound 5 increased the frequency of petite mutants, suggesting an effect on mitochondrial function; however, its retained activity against these mutants and its synergism with fluconazole in the petite background indicate an additional mechanism of action. These findings identify 5 as a promising antifungal adjuvant and support its potential use in combination therapy to enhance azole efficacy against C. glabrata. ImportanceInvasive infections caused by C. glabrata are increasingly hard to treat because this pathogen is often tolerant to azoles and is developing resistance to echinocandins, leaving clinicians with few effective options. Our work explores a nickel-caffeine complex that shows selective activity against C. glabrata and low toxicity, features that make it attractive as a potential therapeutic lead. Notably, this compound enhances the activity of fluconazole, turning a commonly used fungistatic drug into a fungicidal combination against both susceptible and resistant C. glabrata. By also affecting mitochondrial function yet remaining active in respiratory-deficient mutants, the compound appears to act through mechanisms distinct from existing antifungals. These properties suggest that metal-based xanthine complexes could be developed as adjuvants to restore or boost azole efficacy against difficult-to-treat C. glabrata infections, addressing an important priority identified for fungal pathogens.

microbiology↗

bioRxiv2025

Quantitative microinjection of Candida albicans in zebrafish embryos for infection monitoring and survival analysis

This protocol describes a versatile in vivo zebrafish embryo infection model with Candida albicans that enables precise microinjection and monitoring of defined infection readouts to compare experimental conditions (e.g., injection site and incubation temperature). The core workflow consists of quantitative microinjection of DiI-labeled C. albicans into the hindbrain ventricle of 30-48 hpf embryos, incubation at 23 deg C, and daily survival scoring to evaluate condition-dependent mortality. We calibrate the inoculum by hemocytometer counts and by plating the same injection suspension to determine colony-forming units (CFU) per pulse, allowing CFU per embryo to be reported and experiments to be compared across laboratories. Survival is analyzed with Kaplan-Meier curves and, when groups are compared, Cox models. DiI fluorescence verifies successful delivery, excludes mis-injections, and permits semi-quantitative burden comparisons between injection sites and temperatures at 24-48 hpi; beyond [~]48 hpi, signal becomes unreliable due to dye dilution and redistribution. The protocol is designed to be reproducible without transgenic reporters or confocal microscopy and includes practical guidance on randomization, blinding, sample-size planning, and reporting.

microbiology↗

bioRxiv2024

Caspofungin binding to iron compromises its antifungal efficacy against Candida albicans

Echinocandin drugs, such as caspofungin, inhibit the synthesis of {beta}-1,3-D-glucans, which are essential components of the fungal cell wall. These drugs are often the preferred option for treating invasive fungal infections (IFIs) caused by Candida spp. due to their superior efficacy compared to other antifungal agents. Iron overload conditions, which exacerbate fungal burden, are well-documented as significant risk factors for the progression of IFIs. Recent in vitro studies have suggested that iron overload may also reduce the efficacy of cell wall-perturbing agents, such as echinocandins, against Candida albicans, by altering the composition of the fungal cell wall. Here, we show that iron loading conditions which do not interfere with the cell wall composition are still capable of recapitulating the caspofungin-resistant phenotype induced by iron in C. albicans. Spectroscopic analyses provided evidence that caspofungin binds to iron through its ethylenediamine moiety and two amide groups. Consistent with the in vitro activity of {beta}-1,3-D-glucan synthase, molecular dynamics simulations revealed that, when bound to iron, caspofungin undergoes conformational changes that may reduce its ability to inhibit the enzyme. Importantly, the in vivo antifungal efficacy of caspofungin is compromised in a Galleria mellonella model of IFI caused by C. albicans simulating a context of iron overload. This effect may extend beyond C. albicans infections, as the antagonism between iron and caspofungin was also observed in other medically important fungi causing IFIs.

microbiology↗

bioRxiv2021

Copper Acts Synergistically with Fluconazole in Candida glabrata by Compromising Drug Efflux, Sterol Metabolism, and Zinc Homeostasis

The synergistic combinations of drugs are promising strategies to boost the effectiveness of current antifungals and thus prevent the emergence of resistance. In this work, we show that copper and the antifungal fluconazole act synergistically against Candida glabrata, an opportunistic pathogenic yeast intrinsically tolerant to fluconazole. Analyses of the transcriptomic profile of C. glabrata after the combination of copper and fluconazole showed that the expression of the multidrug transporter gene CDR1 was decreased, suggesting that fluconazole efflux could be affected. In agreement, we observed that copper inhibits the transactivation of Pdr1, the transcription regulator of multidrug transporters and leads to the intracellular accumulation of fluconazole. Copper also decreases the transcriptional induction of ergosterol biosynthesis (ERG) genes by fluconazole, which culminates in the accumulation of toxic sterols. Co-treatment of cells with copper and fluconazole should affect the function of proteins located in the plasma membrane, as several ultrastructural alterations, including irregular cell wall and plasma membrane and loss of cell wall integrity, were observed. Finally, we show that the combination of copper and fluconazole downregulates the expression of the gene encoding the zinc-responsive transcription regulator Zap1, which possibly, together with the membrane transporters malfunction, generates zinc depletion. Supplementation with zinc reverts the toxic effect of combining copper with fluconazole, underscoring the importance of this metal in the observed synergistic effect. Overall, this work, while unveiling the molecular basis that supports the use of copper to enhance the effectiveness of fluconazole, paves the way for the development of new metal-based antifungal strategies.

microbiology↗

bioRxiv2021

Impact of Human Serum Proteins on Susceptibility of Acinetobacter baumannii to Cefiderocol: role of iron transport

Cefiderocol is a siderophore antibiotic that co-opts iron transporters to facilitate cell entry. We show that genes related to iron uptake systems and resistance to {beta}-lactams in Acinetobacter baumannii have altered expression levels in the presence of human serum, human serum albumin, or human pleural fluid. Cefiderocol MICs are also raised in the presence of the mentioned fluids. Clinical response in A. baumannii infections may be related to the interplay of these human factors.

microbiology↗

bioRxiv2021

Interplay between meropenem and human serum albumin on expression of carbapenem resistance genes and natural competence in Acinetobacter baumannii

Acinetobacter baumannii A118, a mostly susceptible strain, and AB5075, carbapenem-resistant, were cultured in L broth or L broth with different supplements: 3.5% human serum albumin (HSA), human serum (HS), meropenem, or meropenem plus 3.5% HSA. Natural transformation levels were enhanced in A. baumannii A118 and AB5075 cultured in medium supplemented with 3.5 % HSA. Addition of meropenem plus 3.5% HSA caused synergistic enhancement of natural transformation in A. baumannii A118. Medium containing 3.5% HSA or meropenem enhanced the expression levels of the competence and type IV pilus associated genes. The combination meropenem plus 3.5% HSA produced a synergistic augmentation in the expression levels of many of these genes. The addition of HS, which has a high content of HSA, was also an inducer of these genes. Cultures in medium supplemented with HS or 3.5% HSA also affected resistance genes, which were expressed at higher or lower levels depending on the modification required to enhance resistance. The inducing or repressing activity of these modulators also occurred in three more carbapenem-resistant strains tested. An exception was the A. baumannii AMA16 blaNDM-1 gene, which was repressed in the presence of 3.5% HSA. In conclusion, HSA produces an enhancement of natural transformation and a modification in expression levels of competence genes and antibiotic resistance. Furthermore, when HSA is combined with carbapenems, which may produce stronger cellular stress, the A. baumannii responds increasing the levels of expression of genes involved in natural competence. This process may favor the acquisition of foreign DNA and accelerate evolution. ImportanceAcinetobacter baumannii causes a variety of nosocomial- and community-infections that are usually resistant to multiple antimicrobial agents. As new strains acquire more resistance genes, these infections become harder to treat, and mortality can reach up to 39%. The high genomic plasticity exhibited by A. baumannii must be the consequence of numerous mechanisms that include acquiring foreign DNA and recombination. Here, we describe the ability of A. baumannii to induce competence genes when exposed to environments that resemble those found in the human body during untreated infection or after administration of carbapenems. In this latter scenario expression of genes related to resistance also modify their expression levels such that resistance is increased. The contributions of this article are two-pronged. First, when A. baumannii is exposed to substances present during infection, it responds, augmenting the ability to capture DNA and accelerate evolution. Second, in those conditions, the bacterium also modifies the expression of resistance genes to increase its resistance levels. In summary, recognition of substances that are naturally (HSA) or artificially (treatment with carbapenems) induces A. baumannii to defend, enhancing resistance and increasing the chances of acquiring new resistance mechanisms.

microbiology↗

bioRxiv2020

Cerebrospinal fluid (CSF) boosts metabolism and virulence expression factors in Acinetobacter baumannii

In a recent report by the Centers for Disease Control and Prevention (CDC), multidrug resistant (MDR) Acinetobacter baumannii is a pathogen described as an "urgent threat". Infection with this bacterium manifests as different diseases such as community and nosocomial pneumonia, bloodstream infections, endocarditis, urinary tract, wound infections, burn infections, skin and soft tissue infections, and meningitis. In particular, nosocomial meningitis, a common complication of neurosurgery caused by extensively-drug resistant (XDR) A. baumannii, is extremely challenging to manage. Therefore, it is necessary to identify signals, such as exposure to cerebrospinal fluid (CSF), that trigger expression of virulence factors that are associated with the successful establishment and progress of this infection. While a hypervirulent A. baumannii strain did not show changes in its transcriptome when incubated in the presence of CSF, a low-virulence isolate showed significant differences in gene expression and phenotypic traits. Exposure to 4% CSF caused increased expression of virulence factors such as fimbriae, pilins, and iron chelators, and virulence as determined in various model systems. Furthermore, although CSFs presence did not enhance bacterial growth, it was associated with an increase of expression of genes encoding transcription, translation, and the ATP synthesis machinery. Experiments to identify the active CSF component pointed to human serum albumin (HSA). ImportanceAcinetobacter baumannii, notorious for its multidrug resistant phenotype, overcomes nutrient deprived and desiccated conditions through its metabolic flexibility, pathogenic and physiological adaptability. Although this pathogen is commonly associated with respiratory infections, there have been a considerable amount of cases of A. baumannii bacterial meningitis. These infections are usually post-neurological surgery complications associated with high mortality rates ranging from 40 to 70%. This work describes interactions that may occur during A. baumannii infection of human cerebrospinal fluid (CSF). A. baumanniis displays capabilities to persist and thrive in a nutrient-limited environment, which also triggers the expression of virulence factors. This work also further explores A. baumanniis utilization of an essential component within CSF to trigger enhanced expression of genes associated with its pathoadaptibility in this environment.

microbiology↗