Antifungal heteroresistance has emerged as a clinical challenge across diverse species. In Candida glabrata, however, it has rarely been reported. Here, we characterized heteroresistance in a multicenter collection of 156 C. glabrata isolates, revealing a 25% prevalence with caspofungin specificity and 37°C dependent phenotypes. Transcriptomic profiling of the heteroresistant subpopulation under escalating drug pressure revealed an adaptive program centered on cell cycle and cell wall integrity, including several members of the calcineurin pathway. Mechanically, we identified that the phenotype was abolished by pharmacological inhibition or Δcnb1, Δcrz1 genetic deletion, confirming the regulatory role of calcineurin. Crucially, heteroresistance functioned as a reservoir for resistance, with in vitro descendant strains spanning a spectrum of MIC alterations. Mechanistically, we found the heteroresistance phenotype mostly independent of aneuploidy. To decipher the genetic basis, we applied an unbiased machine-learning framework to genomic data, which not only identified the canonical FKS2_F659del mutation but also prioritized novel candidate PIR2_G149_I167del, demonstrating its power to uncover drivers of resistance from complex datasets. In summary, our study established a stepwise model of heteroresistance in C. glabrata, wherein calcineurin serves as a master regulator that promotes a resistance reservoir, revealing a potential vulnerability that could be exploited to prevent treatment failure.

Antifungal tolerance can promote the emergence of resistance yet often incurs fitness costs for fungal pathogens. How tolerant populations compensate for these deficits and how they may be therapeutically targeted remain poorly understood. Here, we investigate four sequential Candida parapsilosis isolates recovered from a patient with persistent candidemia and failure of micafungin therapy. The infection was ultimately cleared with liposomal amphotericin B (LAMB). Whole-genome sequencing (WGS) confirmed clonal relatedness and the absence of known resistance mutations. Later isolates displayed marked cell wall remodeling (CWR), characterized by increased mannan and reduced β-glucan content, as revealed by microscopy and solid-state nuclear magnetic resonance. These isolates formed thicker biofilms and displayed enhanced echinocandin tolerance but paradoxically showed increased susceptibility to amphotericin B (AMB) in vitro and during systemic infection in mice. Despite a complex mutational landscape, transcriptomic profiling across planktonic and biofilm growth showed minimal divergence from the earliest isolate. Functionally, evolved isolates suppressed M1 macrophage polarization, dampened proinflammatory cytokine production, survived better during neutrophil interactions, and transiently increased fungal burden in vivo. These findings show that host-driven CWR could promote echinocandin tolerance while simultaneously sensitizing C. parapsilosis to AMB. Our results suggest that alternating echinocandin and LAMB therapy may effectively eliminate echinocandin-tolerant fungal populations.IMPORTANCEAntifungal tolerance is increasingly recognized as a precursor to resistance, yet its clinical and biological consequences remain poorly defined. By analyzing sequential Candida parapsilosis isolates from a case of persistent candidemia, we show that cell wall remodeling is associated with echinocandin tolerance, alters host immune interactions, and increases susceptibility to amphotericin B (AMB). These findings reveal how tolerance-associated adaptations shape pathogen fitness during infection and highlight the therapeutic potential of alternating echinocandin and AMB therapy. This work advances our understanding of antifungal tolerance and suggests that exploiting opposing drug susceptibilities may improve treatment outcomes for challenging-to-treat Candida infections.

Transposon mutagenesis coupled with deep sequencing (Tn-seq) is currently being deployed in microbial eukaryotes, including the opportunistic yeast pathogen Candida glabrata, for functional genomics research. This method depends on the generation of highly diverse pools of transposon insertion mutants to cover all genes while minimizing the presence of markers and remnants of engineering. Up to now, pools of Hermes transposon insertion mutants in C. glabrata were generated in uracil-requiring ura3∆ auxotrophs, limiting their use in nutrient-restricted environments, such as those of the host. Indeed, we found that ura3∆ mutants were outcompeted by URA3+ prototrophs during colonization of the mouse gastrointestinal tract. To avoid using auxotrophs in Tn-seq experiments, a new scheme was developed for generating prototrophic pools of Hermes insertion mutants. The scheme involved introducing a recessive cycloheximide resistance mutation in the chromosomal RPL28 gene, which did not alter fitness during mouse colonization. When implemented in several different strains of C. glabrata, high insertion densities were obtained, and differences in subtelomeric chromatin compaction were observed that correlated with natural variation in the silencing gene, SIR3. However, all the strains lacked insertions in the PDR1 and CDR1 genes, which are necessary for resistance to cycloheximide and other antifungals. We directly tested the effect of pdr1∆ mutants and found that they exhibited moderate fitness defects in the gastrointestinal tract of mice even in the absence of antifungals. Thus, the new scheme easily generates high-quality pools of insertion mutants in prototrophic C. glabrata with only minor and knowable limitations. IMPORTANCE:Treatment of fungal infections may be improved by a deeper understanding of the genetic mechanisms of colonization within host organisms. Current approaches to deep genetic sequencing in eukaryotic microbes often involve engineered components that have significant biases, minimize microbial complexity, or alter the normal in vivo fitness of opportunistic fungal pathogens. This study designs a new method for developing transposon insertion mutants in Candida glabrata that does not innately introduce altered fitness in mouse models of gastrointestinal tract infection. This scheme is also portable across strains and possibly even fungal species. The findings show that the new method can be used in this pathogenic yeast to yield highly complex pools and reliably identify genetic components of colonization in mouse models of infection.

A notable increase in azole resistance among Candida tropicalis has been observed worldwide, particularly in the Asia-Pacific region, associated with the expansion of an emerging resistant population named cluster AZR. Here, we present up-to-date epidemiological, antifungal susceptibility and population genomic data from the China Hospital Invasive Fungal Surveillance Net (CHIF-NET) study. A total of 911 C. tropicalis isolates causing invasive candidiasis were collected in 2022-2023, representing 16.1% of all Candida isolates and ranking as the third most common species. High resistance rates to fluconazole (42.3%) were observed, with 87.5% showing cross-resistance to voriconazole. Candidemia isolates showed significantly higher azole resistance than non-candidemia isolates (46.1% vs 39.2%, p = 0.036). Fluconazole resistance rate was higher in medical departments (55.0%) but lower in ICUs (33.7%). Longitudinal analysis demonstrated an overall upward trend in fluconazole resistance from 5.7% since 2009. Although a transient decline was observed during 2020-2021, resistance rebounded thereafter and reached a historical peak of 43.1% in 2022. Population genomic analysis of 604 isolates identified cluster AZR as the predominant fluconazole-resistant population (58.6%), and its expansion was associated with the temporal increase in azole resistance. Of note, all cluster AZR isolates harboured the ERG11 A395 T key resistant mutation, and 98.2% had increased ERG11 copy number. Besides, genomic analysis identified strain replacement and dual-clade mixed infection in cluster AZR-associated clinical cases. In conclusion, the high prevalence of azole resistance in C. tropicalis and the expansion of cluster AZR represent a significant clinical challenge and underscore the need for effective interventions.

Candida parapsilosis is a major human fungal pathogen, with recent global outbreaks driven by fluconazole-resistant (FLCR-Cp) isolates that are difficult to eradicate and associated with poor clinical outcomes. However, the microbial traits enabling persistence of these outbreak lineages remain poorly defined. Here, we show that FLCR-Cp isolates responsible for prolonged, multi-country outbreaks consistently exhibit a striking low-biofilm-producing (LBP) phenotype. Contrary to the prevailing view that robust biofilm formation promotes persistence, LBP strains displayed enhanced stress tolerance, increased cell wall masking, and reduced immune recognition. These traits conferred resistance to neutrophil and macrophage killing and enhanced survival in immune cell-rich organs during systemic infection. Genome-wide transcriptomic profiling revealed extensive metabolic and regulatory rewiring in LBP strains. Whole-genome sequencing (WGS) of a global isolate collection further demonstrated that the LBP phenotype has emerged independently multiple times, supporting convergent evolution under host selection. Functional genomic analyses suggest that biofilm attenuation arises through multigenic changes, and disruption of key biofilm-associated transcriptional regulators enhanced fitness during immune interactions. Together, our findings overturn the assumption that robust biofilm formation drives outbreak persistence and instead identify biofilm attenuation as an adaptive tradeoff that promotes immune evasion and long-term survival. These results redefine our understanding of C. parapsilosis adaptation during healthcare-associated outbreaks and shift attention toward host-driven evolutionary processes than environmental persistence alone.

ABSTRACT Host stresses are often considered a major barrier against the emergence of echinocandin resistance (ECR) in prominent infecting organisms like Candida albicans due to fitness defects. Yet, ECR strains of C. glabrata carrying diverse amino acid changes in Fks1 and Fks2 are increasingly reported as breakthrough infections. Nonetheless, the impact of equivalent mutations in different FKS alleles on fitness has not been systematically studied. Herein, we employed a diverse array of ex vivo and in vivo models to address these questions among clinically relevant ECR mutants. All ECR mutants retained fitness during interaction with THP1 macrophages and neutrophils. Whereas a strain with a Fks2 F659del or fks2Δ showed fitness defects during interaction with macrophages and neutrophils. Fks2 F659del showed a unique susceptibility to numerous stresses, especially the combination of alternative carbon sources, low pH, and H 2 O 2 . Consistent with failure in mounting adaptive oxidative stress response during exposure to H 2 O 2 , transcriptomic analysis of intracellular Fks2 F659del highlighted the dysregulation of oxidative stress response genes, whereas intracellular fks2Δ showed hallmarks of metabolic dysregulation. Intriguingly, the Fks2 F659del mutant was outcompeted by wild type and Fks2 F659V in in vivo gut colonization and systemic infection models. Importantly, whereas both FKS1 and FKS2 were required to establish gut colonization, only FKS2 was required for systemic infection. Therefore, our study supports the notion that the prevalence of ECR mutants among C. glabrata strains is likely driven by its ability to retain fitness across diverse niches. Furthermore, we identified that the essentiality of FKS1 and FKS2 is similarly dictated by niche-specific requirements.

Echinocandins are frontline antifungal drugs, and the emergence of echinocandin-resistant (ECR) species, such as Nakaseomyces glabratus, complicates patient outcomes. Intriguingly, under laboratory conditions, we previously showed that echinocandin alternation with metabolic-independent antifungals, such as amphotericin B (AMB), more effectively kills and minimizes the ECR in N. glabratus. Building upon our previous observations, we examined the efficacy of echinocandin alternation to amphotericin B (EAMB) over echinocandin monotherapy using a systemic candidiasis mouse model to assess if EAMB warrants investigation with potential for clinical evaluation. Interestingly, we show that regardless of the mice's immune status (immunocompromised and immunocompetent) and the N. glabratus isolates [high and low echinocandin tolerance (ECT)] tested, EAMB more rapidly cleared the infection, and minimized ECR in all organs tested compared to caspofungin monotherapy. Pharmacokinetic data suggested that the superiority of EAMB is due to concentration-independent killing activity of liposomal AMB. Although biomarkers suggested higher kidney and liver damage in the EAMB group, histological analysis showed similar damage among both groups. Collectively, using comprehensive ex vivo and in vitro/in vivo experimental conditions, we introduce a novel antifungal therapeutic regimen, which effectively minimizes the ECT and ECR rate in N. glabratus and lays the foundation for in-human studies and clinical trials.

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its impact on public health continue to demand attention as the virus continues to evolve, demonstrating a remarkable ability to adapt to diverse selective pressures including immune responses, therapeutic treatments, and prophylactic interventions. The SARS-CoV-2 variant landscape remains dynamic, with new subvariants continuously emerging, many harboring spike protein mutations linked to immune evasion. In this study, we characterized a panel of live SARS-CoV-2 strains, including those key subvariants implicated in recent waves of infection. Our findings revealed a significant variability in mutation patterns in the spike protein across the strains analyzed. Commercial antibodies and human convalescent plasma (HCoP) samples from unvaccinated donors were ineffective in neutralizing the most recent Omicron subvariants, particularly after the emergence of JN.1 subvariant. Using human airway epithelial cells derived from healthy bronchiolar tissue (hBAEC), we established both monoinfections and coinfections involving SARS-CoV-2, Influenza A virus H1N1 (IFAV_H1N1) and Respiratory Syncytial Virus (RSV). Assessments were conducted to compare viral infectivity and the production and release of immune mediators in the apical and basolateral compartments. Notably, Omicron KP.3.1.1 subvariant induced a more pronounced cytopathic effect in hBAEC compared to its parental strain JN.1 and even surpassed the impact observed with the ancestral wild-type virus (WA1/2020, Washington strain). Furthermore, the coinfection of KP.3.1.1 subvariant with IFAV_H1N1 or RSV did not attenuate SARS-CoV-2 infectivity; instead, it significantly exacerbated the pathogenic synergy in the lung epithelium. Our study demonstrated that pro-inflammatory cytokines IL-6, IFN-β, and IL-10 were upregulated in hBAEC following SARS-CoV-2 monoinfection with recent Omicron subvariants as well as during coinfection with IFAV_H1N1 and RSV. Taken together, our findings offer new insights into the immune evasion strategies and pathogenic potential of evolving SARS-CoV-2 Omicron subvariants, as well as their interactions with other respiratory viruses, carrying important implications for therapeutic development and public health preparedness.

Fungal cell wall-synthesizing enzyme 1,3-β-glucan synthase (GS) is the target of the echinocandins, a frontline antifungal drug class. However, increasing echinocandin resistance due to mutations in GS has been observed in certain fungal pathogens, notably Candida glabrata, where GS is encoded by two homologous genes, FKS1 and FKS2. Despite the importance of GS in the fungal life cycle and as a drug target, the regulation of its expression in culture and in the host is still poorly understood. In this study, we used a fluorescent transcriptional reporter, quantitative reverse-transcriptase PCR, protein analysis, and mining of RNA-seq data sets to examine the regulation of C. glabrata GS expression. We determined that FKS1 and FKS2 promoter activities peak during S-phase and that during exponential growth in culture, FKS1 is expressed at higher levels than FKS2. Interestingly, although FKS2 mRNA expression appeared to be strongly induced in an fks1∆ mutant, this calcineurin-mediated induction was not accompanied by increased FKS2 promoter activity, suggesting post-transcriptional regulation. Examination of FKS2 transcript across the ORF as well as Fks2 protein levels was consistent with post-transcriptional regulation. Finally, RNA-seq data mining revealed that, in contrast to vegetative growth, during the stationary phase and under host conditions, FKS2 is expressed at equivalent or higher levels than FKS1. Together, these experiments revealed that in C. glabrata, the expression of both GS subunits is regulated transcriptionally by the cell cycle, that FKS2 expression is regulated post-transcriptionally by calcineurin and fks1∆, and that in the host, FKS2 may be the more abundant subunit.

Caspofungin is an echinocandin antifungal that inhibits glucan synthesis in the fungal cell wall. A Candida parapsil osis bloodstream isolate resistant to echinocandins was recovered from a patient who had undergone allogeneic hematopoietic stem cell transplantation. The FKS1 gene, encoding the target glucan synthase, contained a heterozygous mutation resulting in an I1380T amino acid change, in addition to the naturally occurring P660A polymorphism. When expressed at the equivalent position in the Fks1p protein of C. lusitaniae , P642A and I1359T, alone and in combination, led to 6-, 12-, and ≥256-fold increases in the minimal inhibitory concentration (MIC) of caspofungin, respectively. The caspofungin concentration needed to inhibit 50% of glucan synthase activity was increased 3-, 37-, and 270-fold, respectively. At high drug concentrations, and also in drug-free medium, infrared spectroscopy revealed a decrease in β-glucan content and an increase in chitin in the cell wall of the I1359T Fks1p mutants. Atomic force microscopy showed cell wall damage and cell swelling in both susceptible and resistant strains under caspofungin exposure. Analysis of susceptibility to cell-wall stressors and key factors in cell wall integrity (CWI) and high-osmolarity glycerol (HOG) pathways showed that all strains activated these pathways under caspofungin stress. In the I1359T Fks1p mutants, Mkc1p was constitutively activated even without caspofungin. Deletion of MKC1 restored caspofungin susceptibility, indicating that activation of the CWI pathway is a key molecular determinant of resistance in vitro to caspofungin in these mutants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a pressing global threat, having emerged in December 2019. Alongside it, Middle East respiratory syndrome coronavirus (MERS-CoV), a highly pathogenic human coronavirus, was identified in 2012 and continues to cause seasonal outbreaks in the Middle East. The persistence of these deadly human coronaviruses underscores the need for ongoing research on broad-spectrum antivirals. Human alveolar A549 cells have been widely used to study respiratory virus infections; however, there is a lack of standardized cell models that are permissive to these diverse lethal coronaviruses. To facilitate the assessment and validation of antiviral treatments, a robust human cell model that is susceptible to SARS-CoV-2, MERS-CoV, and other human coronavirus family members is indispensable. SARS-CoV-2 uses the angiotensin converting enzyme 2 (Ace2) receptor for entry and transmembrane serine protease 2 (Tmprss2) to prime its spike protein, while MERS-CoV relies on the dipeptidyl peptidase 4 receptor (Dpp4) for cellular entry, both of which are minimally expressed in A549 cells. In this study, we fine-tuned the expression levels of each receptor for optimal viral entry and infectivity using lentiviral transduction, cell sorting and clone selection. We successfully developed a robust human cell model expressing multiple viral receptors and demonstrated its susceptibility to both lethal coronaviruses and seasonal human coronaviruses, OC43 and 229E. We also compared two known 3C-like protease inhibitors and found that Nirmatrelvir is superior to Pomotrelvir in terms of pan-coronavirus antiviral activity. Furthermore, we tested 13 known antimalarial drugs and identified Halofantrine as having antiviral activity against SARS-CoV-2. Our findings suggest that this novel human cell model is a valuable and versatile tool for the screening and identification of pan-CoV antiviral drugs.

The persistent evolution of SARS-CoV-2 has led to the emergence of antigenically distinct Omicron subvariants exhibiting increased transmissibility, immune evasion, and altered pathogenicity. Among these, recent subvariants such as JN.1, KP.3.1.1, and LB.1 possess unique antigenic and virological features, underscoring the need for continued surveillance and therapeutic evaluation. As vaccines and commercial monoclonal antibodies show reduced effectiveness against these variants, the role of direct-acting antivirals, such as Nirmatrelvir, targeting conserved viral elements like the main protease inhibitor, becomes increasingly crucial. In this study, we investigated the replication kinetics, host immune responses, and therapeutic susceptibility of three recently circulating Omicron subvariants in the K18-hACE2 transgenic mouse model, using the SARS-CoV-2 parent WA1/2020 strain as a reference. Omicron subvariants exhibited a marked temporal shift in viral infection kinetics characterized by an early lung viral titer peak (~7–8 Log PFU) at 2 days post-infection (dpi), followed by a decline (1–3 Log PFU) by 4 dpi. Pulmonary cytokine and chemokine responses (GM-CSF, TNF-α, IL-1β, IL-6) showed an earlier increase in subvariant-infected mice compared to a gradual response in WA1/2020 infection. Notably, Nirmatrelvir treatment led to significant reductions in lung viral titers in subvariant-infected mice compared to WA1/2020, surpassing its efficacy against the parent strain. These findings highlight that infection with Omicron subvariants yields a broad dynamic range in viral burden with minimum variability, while retaining a prominent therapeutic response to Nirmatrelvir. This study provides insights into the emerging subvariants’ pathogenesis and therapeutic responsiveness, reinforcing the importance of continued variant monitoring and the development of effective countermeasures.

The COVID-19 pandemic has emphasized the necessity for rapid and adaptable drug screening platforms against live pathogenic viruses that require high levels of biosafety containment. Conventional antiviral testing is time-consuming and labor-intensive. Here, we outline the design and validation of a semi-automated drug-screening platform for SARS-CoV-2 that utilizes multiple liquid handlers, a stable A549 cell line expressing ACE2 and TMPRSS2 receptors, and a recombinant SARS-CoV-2 strain harboring the nano-luciferase gene. This platform allows for accelerated low-, mid-, and high-throughput screenings by bypassing the virus inactivation and the staining steps compared to assays utilizing fluorescent reporter viruses or immunofluorescence. First, we demonstrated that the luminescence signal obtained at 24 h post-infection is robust and can be used as a surrogate for fluorescent reporter viruses and immunofluorescence assays that require 48 h incubation post infection. We confirmed the susceptibility of the reporter virus to a panel of reference drugs and validated the luminescence signal in 96- and 384-well plates in accordance with NIH criteria for high-throughput screening. The validation assays showed reproducible results, robust Z factor of ≥0.5, and a coefficient of variation of <20% achieved in both 96 and 384-well plate formats. Lastly, we assessed the assay's performance by screening 240 compounds from the MMV Global Health Library, using the 384-well plate format and remdesivir as a control compound. The single point screening resulted in the identification of 48 hits that inhibited more than 50% of the viral growth. We selected the 15 most active compounds to evaluate their inhibitory concentration and their cytotoxicity, which resulted in the confirmation of the 3 most potent and least toxic compounds that were never reported as antivirals. These results confirm that our platform can be reliably employed for rapid drug screening against SARS-CoV-2 and can be easily adapted to other nano-luciferase reporter viruses.

Candida auris is a significant public health threat due to its environmental persistence and multidrug resistance, with echinocandins being the preferred treatment. However, in addition to resistance, echinocandin tolerance and heteroresistance may contribute to treatment challenges. Echinocandin tolerance involves reduced drug-mediated killing, while heteroresistance is the ability of a small cell subset to grow at high drug concentrations. These phenomena may facilitate the emergence of full resistance and complicate clinical outcomes. The clinical significance of these mechanisms remains unclear, with limited data correlating them with treatment failures. Research is needed to understand their mechanisms and impact, develop streamlined and robust methods to detect them in clinical settings, and explore mitigation strategies. The pathogen's range of drug adaptations demands innovative approaches like spatial transcriptomics to dissect these complex responses and improve patient outcomes.

We investigated the activity of the tuberculosis drug SQ109 against 16 fungal pathogens: Candida albicans, C. auris, C. glabrata, C. guilliermondi, C. kefyr, C. krusei, C. lusitaniae, C. parapsilosis, C. tropicalis, Cryptococcus neoformans, Rhizopus spp., Mucor spp., Fusarium spp., Coccidioides spp., Histoplasma capsulatum and Aspergillus fumigatus. MIC values varied widely (125 ng/mL to >64 μg/mL) but in many cases we found promising (MIC ∼ 4 μg/mL) activity as well as MFC/MIC ratios of ∼ 2. SQ109 metabolites were inactive. The activity of 12 analogs of SQ109 against Saccharomyces cerevisiae correlated with protonophore uncoupling activity, suggesting mitochondrial targeting, consistent with the observation that growth inhibition was rescued by agents which inhibit ROS species accumulation. SQ109 disrupted H+/Ca2+ homeostasis in S. cerevisiae vacuoles, and there was synergy (FICI ∼ 0.26) with pitavastatin, indicating involvement of isoprenoid biosynthesis pathway inhibition. SQ109 is, therefore, a potential antifungal agent with multitarget activity.

Fungal plasma membrane proteins represent key therapeutic targets for antifungal agents, yet their native structure and spatial distribution remain poorly characterized. Herein, we employ an integrative approach to investigate the organization of plasma membrane protein complexes in Candida glabrata, focusing on two abundant and essential membrane proteins, the β-(1,3)-glucan synthase (GS) and the proton pump Pma1. We show that treatment with caspofungin, an echinocandin antifungal that targets GS, disrupts the native distribution of membrane protein complexes and alters membrane biophysical properties. Perturbation of the sphingolipid biosynthesis further modulates drug susceptibility, revealing that the lipid environment plays an integral role in membrane protein organization and GS-echinocandin interactions. Our work highlights the importance of characterizing membrane proteins in their native context to understand their functions and inform the development of novel antifungal therapies.

Macrophages, the central players of innate immunity, control invading microbes by encapsulating them inside the phagosome, a nutrient-poor, reactive oxidant species-rich organelle. Nevertheless, some microbes, including the opportunistic yeast pathogen Candida glabrata , noted for its karyotype diversity, rapid evolution of antifungal drug resistance, and lack of meiosis, can survive and even replicate inside macrophages. However, it is not fully understood how C. glabrata responds to macrophage engulfment, and it is unknown how this presumably DNA-damaging environment influences the pathogen's genome stability. In this study, we used comparative transcriptomics to identify amino acid starvation and DNA damage as conditions eliciting C. glabrata responses most similar to macrophage engulfment. Consistent with this, we found that C. glabrata intra-macrophage survival and replication require master regulator of amino acid biosynthesis GCN4 and functional DNA double-strand break repair. Furthermore, comet assays provided the first direct evidence for increased DNA breaks in intra-macrophage yeast, and pulse-field gel electrophoresis showed that chromosomal alterations occur frequently in macrophage-passaged C. glabrata . Interestingly, these alterations could not be resolved by long read DNA sequencing, suggesting that they involved highly complex repetitive regions. Finally, we identified several point mutations emerging during macrophage passaging and showed that among them, a frameshift in RME1 (repressor of me iosis in Saccharomyces cerevisiae ), increased C. glabrata intra-macrophage fitness. Together, these analyses point to amino acid deprivation, reveal elevated DNA breakage and chromosome instability, and raise intriguing questions about the role of meiotic gene orthologs in C. glabrata persisting and replicating within macrophages.

2583 Background: Epstein−Barr virus (EBV) and human papillomavirus (HPV) are considered human oncoviruses. In contrast, the torque teno virus (TTV) is not associated with any disease but its detection in circulation is associated with the status of the immune system. In this study, we examine the prevalence of active EBV, HPV and TTV viral RNA in patients treated for solid tumors or hematologic neoplasms. In addition, we compared differential expression of selected immune and inflammatory biomarkers between Virus positive (V+) and Virus negative (V-) cases using peripheral blood cell-free RNA (cfRNA). Methods: cfRNA was extracted from the peripheral blood of 581patients with a diagnosis of hematologic neoplasms and 558 patients with solid tumor. cfRNA was sequenced by NGS using a targeted RNA panel of 1600 genes and the viral RNA of TTV, EBV and HPV. Two thirds of the samples were used for training and one third for testing machine learning (ML) system (Bayesian/Random Forest) and exploring the presence of specific inflammatory profiles distinguishing V+ from V- patients. Results: RNA testing was selected to ensure that only active and proliferating viruses were detected. We detected TTV in 52/1139 (4.6%), EBV in 251/1139 (22%), and HPV in 68/1139 (6.0%). TTV with EBV codetection was observed in 11 samples (1%), and with HPV in 4 patients (0.4%). Co-detection of EBV with HPV was observed in 13 patients (1.1%). Using 90 biomarkers in ML algorithm can reliably distinguish V+ from V- with AUC of 0.725 (CI: 0.658-0.791) in the testing set. Significantly higher levels of B-cell markers are noted in V+ patients. PD-L1 mRNA was significantly (P <0.001) higher in V+ patients, which suggests that these patients may be more responsive to checkpoint immune therapy. CD70 is also detected at high level in V+ patients (P<0.0001). Upon comparing between the V+ groups (TTV, EBV, and HPV), there was no statistical difference between the three groups after adjusting for multiple testing. However, some difference in cytokine levels was noted between TTV-positive patients and HPV-positive patients. CD36, IFNA2 and IL17A were higher in TTV-positive cases as compared with HPV-positive cases (P-value 0.0003, 0.005 and 0.004, respectively). Conclusions: Globally, detectable active viruses in plasma of patients with cancer is relatively high (29%) and this detection is associated with a specific immune/inflammatory “activation” signature characterized by transcriptomic upregulation of PD-L1 and CD70 and increase in B-cells. There is no specific signature that distinguishes between the V+ subgroups [TTV vs EBV vs HPV]. However, transcriptionally, CD36, IFNA2 and IL17A upregulation distinguished TTV+ and HPV+ cases, a phenomenon that may indicate HPV ability to initiate an immunosuppressive tumor microenvironment.

Background The widespread emergence of antibiotic resistance including MDR in Gram-negative bacterial pathogens poses a critical challenge to the current antimicrobial armamentarium.Objectives To create a novel drug-Fc conjugate (DFC) that can be delivered at sustained and prolonged levels while simultaneously activating the host immune response to combat MDR Gram-negative infections.Methods The Cloudbreak (TM) platform was used to develop DFCs consisting of a targeting moiety (TM) (a polymyxin-derived dimer) attached via a non-cleavable linker to an effector moiety (EM) (the Fc domain of human IgG1). In vitro activities of the DFCs were assessed by MIC testing. Neutropenic mouse models of thigh infection, septicaemia and pneumonia were used to evaluate in vivo efficacy. Pharmacokinetics were evaluated in mice and cynomolgus monkeys.Results A single prophylactic dose of our lead DFC, CTC-177, resulted in significantly decreased bacterial burdens and reduced inflammation comparable to daily treatment with colistin in septicaemia and pneumonia mouse models. Furthermore, CTC-177 prophylaxis was able to restore colistin efficacy in colistin-resistant septicaemia, reducing bacterial burdens beyond the limit of detection. Finally, CTC-177 displayed a long terminal half-life of over 24 and 65 h in mice and cynomolgus monkeys, respectively.Conclusions These data support the continued development of Cloudbreak (TM) DFCs as broad-spectrum prophylactic agents against Gram-negative infections.