Biology subjects

Santiago-Tirado, F. H.

Publications and source records attributed to Santiago-Tirado, F. H..

7 recordsLinked to original sources

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↗

bioRxiv2024

Lack of an atypical PDR transporter generates an immunogenic Cryptococcus neoformans strain that drives a dysregulated and lethal immune response in murine lungs

Cryptococcus neoformans is an opportunistic fungal pathogen responsible for >150,000 deaths every year with a mortality rate as high as 81%. This high medical burden is due, in part, to an incomplete understanding of its pathogenesis. In a previous study, we identified a cryptococcal atypical ATP-binding cassette (ABC) pleiotropic drug resistance (PDR) transporter, PDR6, that affected antifungal resistance and host interactions. Here, we follow-up on the role of PDR6 in cryptococcal virulence. In vivo, mice infected with the pdr6{Delta} strain display altered symptomatology and disease progression. Specifically, we observed a significant increase in the innate immune cell populations in the pdr6{Delta}-infected mice when compared to their WT-infected littermates. Furthermore, quantification of pulmonary cytokines/chemokines revealed a robust increase of pro-inflammatory cytokines in mice infected with the pdr6{Delta} mutant strain. Despite the documented sensitivity of the pdr6{Delta} strain to azole antifungal drugs, the treatment of pdr6{Delta}-infected animals with antifungals did not affect survival, yet treatment with a corticosteroid significantly extended survival, highlighting the importance of a balanced/controlled host immune response. Results with mice that mount opposing immune responses supports out hypothesis that the pdr6{Delta} strain induces a hyper-inflammatory immune response, and that the mice succumb to immune-dependent tissue damage rather than the fungal burden. This altered immune response is driven, in part, by changes in the mutants surface. Taken together, this study provides insights regarding cryptococcal pathogenesis and highlights additional functions of PDR-type ABC transporters in pathogenic fungi. IMPORTANCEYeasts of the Cryptococcus genus, especially C. neoformans, can cause disease with unacceptably high mortality. This is due to delays in diagnostics, ineffective treatments, and an incomplete understanding of the interactions between this fungus and our immune system. In this study, we expand our knowledge of the biological function of the PDR6 gene, particularly its effect on modulating the hosts immune response. Normally, C. neoformanss infections are characterized by an anti-inflammatory response that is unable to control the yeast. In the absence of PDR6, the response to the infection is a dysregulated pro-inflammatory response that initially controls the fungi but eventually results in death of the host due to too much tissue damage. This is due, in part, to an altered fungal surface. Given the dual role of PDR6 in modulating antifungal sensitivity and immune responses, this work provides important insights that may lead to new or improved therapeutics.

microbiology↗

bioRxiv2023

Phenotypic characterization of HAM1, a novel mating regulator of the fungal pathogen Cryptococcus neoformans

Cryptococcus neoformans is a fungal pathogen responsible for >200,000 yearly cases with a mortality as high as 81%. This burden results, in part, from an incomplete understanding of its pathogenesis and ineffective antifungal treatments; hence, there is a pressing need to understand the biology and host interactions of this yeast to develop improved treatments. Protein palmitoylation is important for cryptococcal virulence, and we previously identified the substrates of its main palmitoyl transferase. One of them was encoded by the uncharacterized gene CNAG_02129. In the filamentous fungus Neurospora crassa, a homolog of this gene named HAM-13 plays a role in proper cellular communication and filament fusion. In Cryptococcus, cellular communication is essential during mating, therefore we hypothesized that CNAG_02129, which we named HAM1, may play a role in mating. We found that ham1{Delta} mutants produce more fusion products during mating, filament more robustly, and exhibit competitive fitness defects under mating and non-mating conditions. Additionally, we found several differences with the major virulence factor, the polysaccharide capsule, that may affect virulence, consistent with prior studies linking virulence to mating. We observed that ham1{Delta} mutants have decreased capsule attachment and transfer but exhibit higher amounts of exopolysaccharide shedding and biofilm production. Lastly, HAM1 expression is significantly lower in mating media relative to non-mating conditions, consistent with it acting as a negative regulator of mating. Understanding the connection between mating and virulence in C. neoformans may open new avenues of investigation into ways to improve the treatment of this disease. ImportanceFungal mating is a vital part of the lifecycle of the pathogenic yeast Cryptococcus neoformans. More than just ensuring the propagation of the species, mating allows for sexual reproduction to occur and generates genetic diversity as well as infectious propagules that can invade mammalian hosts. Despite its importance in the biology of this pathogen, we still do not know all of the major players regulating the mating process and if they are involved or impact its pathogenesis. Here we identified a novel negative regulator of mating that also affects certain cellular characteristics known to be important for virulence. This gene, which we call HAM1, is widely conserved across the cryptococcal family as well as in many pathogenic fungal species. This study will open new avenues of exploration regarding the function of uncharacterized but conserved genes in a variety of pathogenic fungal species, and specifically in serotype A of C. neoformans.

microbiology↗

bioRxiv2022

Real-time visualization of phagosomal pH manipulation by Cryptococcus neoformans in an immune signal-dependent way

Understanding of how intracellular pathogens survive in their host cells is important to improve management of their diseases. This has been fruitful for intracellular bacteria but it is an understudied area in fungal pathogens. Here we start elucidating and characterizing the strategies used by one of the commonest fungal pathogens, Cryptococcus neoformans, to survive intracellularly. The ability of the fungus to survive inside host cells is one of the main drivers of disease progression, yet it is unclear whether C. neoformans resides in a fully acidified, partially acidic, or neutral phagosome. Using a dye that only fluoresce under acidic conditions to stain C. neoformans, a hypha-defective Candida albicans mutant, and the nonpathogenic Saccharomyces cerevisiae, we characterized the fungal behaviors in infected macrophages by live microscopy. The main behavior in the C. albicans mutant strain and S. cerevisiae-phagosomes was rapid acidification after internalization, which remained for the duration of the imaging. In contrast, a significant number of C. neoformans-phagosomes exhibited alternative behaviors distinct from the normal phagosomal maturation: some phagosomes acidified with subsequent loss of acidification, and other phagosomes never acidified. Moreover, the frequency of these behaviors was affected by the immune status of the host cell. We applied the same technique to a flow cytometry analysis and found that a substantial percentage of C. neoformans-phagosomes showed impaired acidification, whereas almost 100% of the S. cerevisiae-phagosomes acidify. Lastly, using a membrane-damage reporter, we show phagosome permeabilization correlates with acidification alterations, but it is not the only strategy that C. neoformans uses to manipulate phagosomal acidification. The different behaviors described here provide an explanation to the confounding literature regarding cryptococcal-phagosome acidification and the methods can be applied to study other intracellular fungal pathogens.

microbiology↗

bioRxiv2022

The ER protein translocation channel subunit Sbh1 controls virulence of Cryptococcus neoformans

The fungal pathogen Cryptococcus neoformans is distinguished by a cell wall-anchored polysaccharide capsule that is critical for virulence. Biogenesis of both cell wall and capsule relies on the secretory pathway. Protein secretion begins with polypeptide translocation across the endoplasmic reticulum (ER) membrane through a highly conserved channel formed by three proteins: Sec61, Sbh1, and Sss1. Sbh1, the most divergent, contains multiple phosphorylation sites, which may allow it to regulate entry into the secretory pathway in a species- and protein-specific manner. Absence of SBH1 causes a cell-wall defect in both Saccharomyces cerevisiae and C. neoformans, although other phenotypes differ. Notably, proteomic analysis showed that when cryptococci are grown in conditions that mimic aspects of the mammalian host environment (tissue culture medium, 37 {degrees}C, 5% CO2), a set of secretory and transmembrane proteins is upregulated in wild-type, but not in{Delta} sbh1 mutant cells. The Sbh1-dependent proteins show specific features of their ER targeting sequences that likely cause them to transit less efficiently into the secretory pathway. Many also act in cell-wall biogenesis, while several are known virulence factors; consistent with these observations, the C. neoformans {Delta}sbh1 mutant is avirulent in a mouse infection model. We conclude that, in the context of conditions encountered during infection, Sbh1 controls the entry of virulence factors into the secretory pathway of C. neoformans, and thereby regulates fungal pathogenicity. ImportanceCryptococcus neoformans is a yeast that causes almost 200,000 deaths worldwide each year, mainly of immunocompromised individuals. The surface structures of this pathogen, a protective cell wall surrounded by a polysaccharide capsule, are made and maintained by proteins that are synthesized inside the cell and travel outwards through the secretory pathway. A protein called Sbh1 is part of the machinery that determines which polypeptides enter this export pathway. We found that when Sbh1 is absent, both C. neoformans and the model yeast S. cerevisiae show cell wall defects. Lack of Sbh1 also changes the pattern of secretion of both transmembrane and soluble proteins, in a manner that depends on characteristics of their sequences. Notably, multiple proteins that are normally upregulated in conditions similar to those encountered during infection, including several needed for cryptococcal virulence, are no longer increased. Sbh1 thereby regulates the ability of this important pathogen to cause disease.

microbiology↗

bioRxiv2022

An atypical ABC transporter is involved in antifungal resistance and host interactions in the pathogenic fungus Cryptococcus neoformans

ATP-binding cassette (ABC) transporters represent one of the largest protein superfamilies. Functionally diverse, ABC transporters have been implicated in many aspects of microbial physiology. The genome of the human fungal pathogen Cryptococcus neoformans encodes 54 putative ABC transporters and the majority of them remain uncharacterized. In a previous genetic screen for fungal regulators of phagocytosis, we identified an uncharacterized gene, CNAG_06909, that modulates host interactions. This gene encodes a half-size ABC transporter of the PDR-type, and phenotypic studies of a strain with this gene deleted revealed an altered antifungal susceptibility profile, including hypersensitivity to fluconazole (FLC). This gene, which we have named PDR6, localizes to the endoplasmic reticulum (ER) and plasma membrane (PM), and when absent, less ergosterol is observed in the PM. Additionally, we observed that the pdr6{Delta} strain displays a reduction in secreted polysaccharide capsular material. These changes to the cellular surface may explain the observed increased uptake by macrophages and the reduced intracellular survival. Finally, studies in mice demonstrate that Pdr6 function is required for normal progression of cryptococcal infection. Taken together, this study demonstrates a novel dual role for PDR transporters in C. neoformans, which could represent a potential target for antifungal therapeutics. Furthermore, the atypical half-size transporter encoded by PDR6 is conserved in many fungal pathogens, but absent in model non-pathogenic fungi. Hence, this study provides for the first time, a function for this unique group of fungal half-size PDR transporters that, although conserved, remain largely understudied. IMPORTANCEConserved across all kingdoms of life, ABC transporters comprise one of the largest protein families. They are associated with multidrug resistance, affecting aspects such as resistance to antimicrobials or anti-cancer drugs. Despite their importance, they are understudied in fungal pathogens. In the environmental fungus Cryptococcus neoformans, a leading cause of fungal infections, only a few ABC transporters have been studied. Here we characterize an atypical, half-size, ABC transporter of the PDR-type, that affects both antifungal resistance and host-pathogen interactions. PDR-type transporters are only present in fungi and plants, and this subgroup of half-size transporters is conserved in fungal pathogens, yet their function was completely unknown. Because the current treatments for cryptococcal infection are suboptimal, understanding the mechanisms of antifungal resistance and the host interactions that drive the infection is critical to improve the management of this disease. Here we provide insights into these important aspects of cryptococcal pathogenesis.

microbiology↗

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↗