Ifu5, a WW domain-containing protein interacts with Efg1 to achieve coordination of normoxic and hypoxic functions to influence pathogenicity traits in Candida albicans.

Hypoxic adaptation pathways, essential for Candida albicans pathogenesis, are tied to its transition from a commensal to a pathogen. Herein, we identify a WW domain-containing protein, Ifu5, as a determinant of hypoxic adaptation that also impacts normoxic responses in this fungus. Ifu5 activity supports glycosylation homeostasis via the Cek1 mitogen-activated protein kinase-dependent up-regulation of PMT1, under normoxia. Transcriptome analysis of ifu5Δ/Δ under normoxia shows a significant up-regulation of the hypoxic regulator EFG1 and EFG1-dependent genes. We demonstrate physical interaction between Ifu5 by virtue of its WW domain and Efg1 that represses EFG1 expression under normoxia. This interaction is lost under hypoxic growth conditions, relieving EFG1 repression. Hypoxic adaptation processes such as filamentation and biofilm formation are affected in ifu5Δ/Δ cells revealing the role of Ifu5 in hypoxic signalling and modulating pathogenicity traits of C. albicans under varied oxygen conditions. Additionally, the WW domain of Ifu5 facilitates its role in hypoxic adaptation, revealing the importance of this domain in providing a platform to integrate various cellular processes. These data forge a relationship between Efg1 and Ifu5 that fosters the role of Ifu5 in hypoxic adaptation thus illuminating novel strategies to undermine the growth of C. albicans.

The 5' Untranslated Region of the EFG1 Transcript Promotes Its Translation To Regulate Hyphal Morphogenesis in Candida albicans.

Extensive 5' untranslated regions (UTR) are a hallmark of transcripts determining hyphal morphogenesis in Candida albicans The major transcripts of the EFG1 gene, which are responsible for cellular morphogenesis and metabolism, contain a 5' UTR of up to 1,170 nucleotides (nt). Deletion analyses of the 5' UTR revealed a 218-nt sequence that is required for production of the Efg1 protein and its functions in filamentation, without lowering the level and integrity of the EFG1 transcript. Polysomal analyses revealed that the 218-nt 5' UTR sequence is required for efficient translation of the Efg1 protein. Replacement of the EFG1 open reading frame (ORF) by the heterologous reporter gene CaCBGluc confirmed the positive regulatory importance of the identified 5' UTR sequence. In contrast to other reported transcripts containing extensive 5' UTR sequences, these results indicate the positive translational function of the 5' UTR sequence in the EFG1 transcript, which is observed in the context of the native EFG1 promoter. It is proposed that the 5' UTR recruits regulatory factors, possibly during emergence of the native transcript, which aid in translation of the EFG1 transcript.IMPORTANCE Many of the virulence traits that make Candida albicans an important human fungal pathogen are regulated on a transcriptional level. Here, we report an important regulatory contribution of translation, which is exerted by the extensive 5' untranslated regulatory sequence (5' UTR) of the transcript for the protein Efg1, which determines growth, metabolism, and filamentation in the fungus. The presence of the 5' UTR is required for efficient translation of Efg1, to promote filamentation. Because transcripts for many relevant regulators contain extensive 5' UTR sequences, it appears that the virulence of C. albicans depends on the combination of transcriptional and translational regulatory mechanisms.

Lipid Signaling via Pkh1/2 Regulates Fungal CO2 Sensing through the Kinase Sch9.

Adaptation to alternating CO2 concentrations is crucial for all organisms. Carbonic anhydrases-metalloenzymes that have been found in all domains of life-enable fixation of scarce CO2 by accelerating its conversion to bicarbonate and ensure maintenance of cellular metabolism. In fungi and other eukaryotes, the carbonic anhydrase Nce103 has been shown to be essential for growth in air (~0.04% CO2). Expression of NCE103 is regulated in response to CO2 availability. In Saccharomyces cerevisiae, NCE103 is activated by the transcription factor ScCst6, and in Candida albicans and Candida glabrata, it is activated by its homologues CaRca1 and CgRca1, respectively. To identify the kinase controlling Cst6/Rca1, we screened an S. cerevisiae kinase/phosphatase mutant library for the ability to regulate NCE103 in a CO2-dependent manner. We identified ScSch9 as a potential ScCst6-specific kinase, as the sch9Δ mutant strain showed deregulated NCE103 expression on the RNA and protein levels. Immunoprecipitation revealed the binding capabilities of both proteins, and detection of ScCst6 phosphorylation by ScSch9 in vitro confirmed Sch9 as the Cst6 kinase. We could show that CO2-dependent activation of Sch9, which is part of a kinase cascade, is mediated by lipid/Pkh1/2 signaling but not TORC1. Finally, we tested conservation of the identified regulatory cascade in the pathogenic yeast species C. albicans and C. glabrata Deletion of SCH9 homologues of both species impaired CO2-dependent regulation of NCE103 expression, which indicates a conservation of the CO2 adaptation mechanism among yeasts. Thus, Sch9 is a Cst6/Rca1 kinase that links CO2 adaptation to lipid signaling via Pkh1/2 in fungi. IMPORTANCE: All living organisms have to cope with alternating CO2 concentrations as CO2 levels range from very low in the atmosphere (0.04%) to high (5% and more) in other niches, including the human body. In fungi, CO2 is sensed via two pathways. The first regulates virulence in pathogenic yeast by direct activation of adenylyl cyclase. The second pathway, although playing a fundamental role in fungal metabolism, is much less understood. Here the transcription factor Cst6/Rca1 controls carbon homeostasis by regulating carbonic anhydrase expression. Upstream signaling in this pathway remains elusive. We identify Sch9 as the kinase controlling Cst6/Rca1 activity in yeast and demonstrate that this pathway is conserved in pathogenic yeast species, which highlights identified key players as potential pharmacological targets. Furthermore, we provide a direct link between adaptation to changing CO2 conditions and lipid/Pkh1/2 signaling in yeast, thus establishing a new signaling cascade central to metabolic adaptation.

Dom34 Links Translation to Protein O-mannosylation.

In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5'-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation. Polysomal profiling revealed that Dom34 stimulates general translation moderately, but that it is especially required for translation of transcripts encoding Pmt isoforms 1, 4 and 6. Because defective protein N- or O-glycosylation upregulates transcription of PMT genes, it appears that Dom34-mediated specific translational upregulation of the PMT transcripts optimizes cellular responses to glycostress. Its translational function as an RNA binding protein acting at the 5'-UTR of specific transcripts adds another facet to the known ribosome-releasing functions of Dom34 at the 3'-UTR of transcripts.

Candida albicans responds to glycostructure damage by Ace2-mediated feedback regulation of Cek1 signaling.

Candida albicans uses the Cek1 MAPK pathway to restore cells from damage of its cell wall glycostructures. Defective protein N- or O-glycosylation activates Cek1 and the transcription factor Ace2 as its downstream target, to upregulate genes encoding protein O-mannosyltransferases (Pmt proteins). In unstressed cells, Cek1-Ace2 activity blocks expression of PMT1, which is de-repressed by tunicamycin. Genomic binding targets of Ace2 included ZCF21, which was upregulated by Ace2 and found to repress PMT1 transcription in unstressed cells. Surprisingly, genes encoding components of the Cek1 pathway including MSB2, CST20, HST7, CEK1 and ACE2 were also identified as Ace2 targets indicating Ace2-mediated transcriptional amplification of pathway genes under N-glycosylation stress. In this condition, physical interaction of the Ace2 protein with the upstream MAPKKK Cst20 was detected. Cst20-GFP showed stress-induced import from the cytoplasm into the nucleus and phosphorylation of Ace2. Interestingly, forced nuclear localization of Cst20 inhibited Cek1-Ace2 signaling, while forced cytoplasmic localization of Cst20 retained full signaling activity, suggesting that nuclear Cst20 downregulates the Cek1 pathway. Collectively, the results indicate that Ace2 is a versatile multifunctional transcriptional regulator, which activates glycostress responses of C. albicans by both positive forward and negative feedback regulation of Cek1 signaling.

Candida utilis and Cyberlindnera (Pichia) jadinii: yeast relatives with expanding applications.

The yeast Candida utilis is used as a food additive and as a host for heterologous gene expression to produce various metabolites and proteins. Reliable protocols for intracellular production of recombinant proteins are available for C. utilis and have now been expanded to secrete proteins into the growth medium or to achieve surface display by linkage to a cell wall protein. A recombinant C. utilis strain was recently shown to induce oral tolerance in a mouse model of multiple sclerosis suggesting future applications in autoimmune therapy. Whole genome sequencing of C. utilis and its presumed parent Cyberlindnera (Pichia) jadinii demonstrated different ploidy but high sequence identity, consistent with identical recombinant technologies for both yeasts. C. jadinii was recently described as an antagonist to the important human fungal pathogen Candida albicans suggesting its use as a probiotic agent. The review summarizes the status of recombinant protein production in C. utilis, as well as current and future biotechnological and medical applications of C. utilis and C. jadinii.

Click beetle luciferases as dual reporters of gene expression in Candida albicans.

Synthetic genes encoding functional luciferases of the click beetle (CB) Pyrophorus plagiophthalamus have been expressed in the human fungal pathogen Candida albicans. Both green- and red-emitting CB luciferases (CaCBGluc and CaCBRluc) were produced with high efficiency in transformants under transcriptional control of the growth-dependent ACT1 promoter, as well as by the HWP1 and UME6 promoters, which are upregulated during hyphal morphogenesis, as well as by the YWP1 and EFG1 promoters, which are downregulated. For all hyphally regulated genes, relative bioluminescence values derived from promoter fusions approximated relative transcript levels of native genes, although downregulation of YWP1 promoter activity required correction for the stability of CB luciferases (approximate half-lives 30 min for CaCBRluc and 80 min for CaCBGluc, as determined by immunoblotting). Importantly, the activity of both luciferases could be separately monitored in a single strain, in intact cells, in lysed cells or in cell extracts using luciferin as single substrate and inhibition of hypha formation by farnesol could be easily detected by the HWP1p-CaCBRluc fusion. The results suggest that CB luciferases are convenient tools to measure gene expression in C. albicans and may facilitate screenings for antifungal compounds.

Oral Tolerance Induction in Experimental Autoimmune Encephalomyelitis with Candida utilis Expressing the Immunogenic MOG35-55 Peptide.

Multiple sclerosis (MS) is an autoimmune disease that attacks myelinated axons in the central nervous system. Induction of oral tolerance is a potent mechanism to prevent autoimmunity. The food yeast Candida utilis was used to test the therapeutic potential of oral tolerance induction in an animal model of human multiple sclerosis (MS). We constructed a C. utilis strain, which displays a fusion peptide composed of the encephalitogenic MOG35-55 peptide and the C. utilis Gas1 cell wall protein on its surface.By immunizing mice with MOG35-55 peptide experimental autoimmune encephalomyelitis (EAE) was induced in a mouse model. Feeding of mice with C. utilis that expresses MOG35-55 peptide on its surface was started seven days prior to immunization and was continued for ten days. Control animals were treated with wild-type fungus or left untreated. Untreated mice developed first clinical symptoms ten days post immunization (p. i.) with an ascending paralysis reaching maximal clinical disability at day 18 to 20 p. i.. Treatment with the wild-type strain demonstrated comparable clinical symptoms. In contrast, oral gavage of MOG35-55-presenting fungus ameliorated the development of EAE. In addition, incidence as well as maximal clinical disease severity were significantly reduced. Interestingly, reduction of disease severity also occurred in animals treated with heat-inactivated C. utilis cells indicating that tolerance induction was independent of fungal viability. Better disease outcome correlated with reduced demyelination and cellular inflammation in the spinal cord, lower T cell proliferation against rechallenge with MOG35-55 and more regulatory T cells in the lymph nodes. Our data demonstrate successful that using the food approved fungus C. utilis presenting the immunogenic MOG35-55 peptide on its surface induced an oral tolerance against this epitope in EAE. Further studies will reveal the nature and extent of an anti-inflammatory environment established by the treatment that prevents the development of an autoimmune disorder affecting the CNS.

Oxygen-independent FbFP: Fluorescent sentinel and oxygen sensor component in Saccharomyces cerevisiae and Candida albicans.

FMN-binding fluorescent proteins (FbFPs) outperform GFP and its derivatives because of their oxygen-independence, small size and rapid maturation. FbFPs have been used successfully as reliable reporters of gene expression in the cytoplasm of pro- and eukaryotes. Here we extend previous findings on the codon-adapted CaFbFP variant, which functions in the apathogenic yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans. In both fungal species, CaFbFP could be targeted to the nucleus and the cell wall by endogenous signals (H2B-/Aga2-fusions) demonstrating its use as a fluorescent beacon in these relevant cellular locations. Transformants of both fungal species producing a CaFbFP-YFP fusion (YFOS) showed variable energy transfer from CaFbFP to YFP (FRET) that depended in its extent on external O2 concentrations. Applications as fluorescent sentinel and oxygen biosensor expand the FbFP toolbox to study oxygen-independent cellular processes under hypoxia.

Hypoxia and Temperature Regulated Morphogenesis in Candida albicans.

Candida albicans is a common commensal in the human gut but in predisposed patients it can become an important human fungal pathogen. As a commensal, C. albicans adapts to low-oxygen conditions and represses its hyphal development by the transcription factor Efg1, which under normoxia activates filamentation. The repressive hypoxic but not the normoxic function of Efg1 required its unmodified N-terminus, was prevented by phosphomimetic residues at normoxic phosphorylation sites T179 and T206 and occurred only at temperatures ≤35°C. Genome-wide binding sites for native Efg1 identified 300 hypoxia-specific target genes, which overlapped partially with hypoxic binding sites for Ace2, a known positive regulator of hypoxic filamentation. Transcriptional analyses revealed that EFG1, ACE2 and their identified targets BCR1 and BRG1 encode an interconnected regulatory hub, in which Efg1/Bcr1 act as negative and Ace2/Brg1 act as positive regulators of gene expression under hypoxia. In this circuit, the hypoxic function of Ace2 was stimulated by elevated CO2 levels. The hyperfilamentous phenotype of efg1 and bcr1 mutants depended on Ace2/Brg1 regulators and required increased expression of genes encoding Cek1 MAP kinase and its downstream target Cph1. The intricate temperature-dependent regulatory mechanisms under hypoxia suggest that C. albicans restricts hyphal morphogenesis in oxygen-poor body niches, possibly to persist as a commensal in the human host.

The structure of the Cyberlindnera jadinii genome and its relation to Candida utilis analyzed by the occurrence of single nucleotide polymorphisms.

The yeast Cyberlindnera jadinii is a close relative of Candida utilis that is being used in the food and feed industries. Here, we present the 12.7Mb genome sequence of C. jadinii strain CBS 1600 generated by next generation sequencing. The deduced draft genome sequence consists of seven large scaffolds analogous to the seven largest chromosomes of C. utilis. An automated annotation of the C. jadinii genome identified 6147 protein-coding sequences. The level of ploidy for both genomes was analyzed by calling single nucleotide polymorphisms (SNPs) and was verified measuring nuclear DNA contents by florescence activated cell sorting (FACS). Both analyses determined the level of ploidy to diploid for C. jadinii and to triploid for C. utilis. However, SNP calling for C. jadinii also identified scaffold regions that seem to be haploid, triploid or tetraploid.

Secreted xylanase XynA mediates utilization of xylan as sole carbon source in Candida utilis.

The fodder yeast Candida utilis is able to use xylose mono- and oligomers as sources of carbon but not the abundant polymer xylan. C. utilis transformants producing the Penicillium simplicissimum xylanase XynA were constructed using expression vectors encoding fusions of the Saccharomyces cerevisiae Mfα1 pre-pro secretion leader to XynA. The Mfα1-XynA fusion was efficiently processed in transformants and XynA was secreted almost quantitatively into the culture medium. Secreted XynA was enzymatically active and allowed transformants to grow on xylan as the sole carbon source. Addition of a second expression unit for the heterologous green fluorescent protein (GFP) generated C. utilis transformants, which showed intracellular GFP fluorescence during growth on xylan. The results suggest that xylanase-producing C. utilis is suited as a cost-effective host organism for heterologous protein production and for other biotechnical applications.

Signaling domains of mucin Msb2 in Candida albicans.

Candida albicans adapts to the human host by environmental sensing using the Msb2 signal mucin, which regulates fungal morphogenesis and resistance characteristics. Msb2 is anchored within the cytoplasmic membrane by a single transmembrane (TM) region dividing it into a large N-terminal exodomain, which is shed, and a small cytoplasmic domain. Analyses of strains carrying deleted Msb2 variants revealed an exodomain segment required for cleavage, shedding, and all functions of Msb2. Phosphorylation of the mitogen-activated protein kinase (MAP kinase) Cek1 was regulated by three distinct regions in Msb2: in unstressed cells, N-terminal sequences repressed phosphorylation, while its induction under cell wall stress required the cytoplasmic tail (C-tail) and sequences N-terminally flanking the TM region, downstream of the proposed cleavage site. Within the latter Msb2 region, overlapping but not identical sequences were also required for hyphal morphogenesis, basal resistance to antifungals, and, in unstressed cells, downregulation of the PMT1 transcript, encoding protein O-mannosyltransferase-1. Deletion of two-thirds of the exodomain generated a truncated Msb2 variant with a striking ability to induce hyperfilamentous growth, which depended on the presence of the Msb2-interacting protein Sho1, the MAP kinase Cek1, and the Efg1 transcription factor. Under cell wall stress, the cytoplasmic tail relocalized partially to the nucleus and contributed to regulation of 117 genes, as revealed by transcriptomic analyses. Genes regulated by the C-tail contained binding sites for the Ace2 and Azf1 transcription factors and included the ALS cell wall genes. We concluded that Msb2 fulfills its numerous functions by employing functional domains that are distributed over its entire length.

A surprising role for the Sch9 protein kinase in chromosome segregation in Candida albicans.

The AGC kinase Sch9 regulates filamentation in Candida albicans. Here, we show that Sch9 binding is most enriched at the centromeres in C. albicans, but not in Saccharomyces cerevisiae. Deletion of CaSch9 leads to a 150- to 750-fold increase in chromosome loss. Thus, we report a previously unknown role of Sch9 in chromosome segregation.

Interplay between Candida albicans and the antimicrobial peptide armory.

Antimicrobial peptides (AMPs) are key elements of innate immunity, which can directly kill multiple bacterial, viral, and fungal pathogens. The medically important fungus Candida albicans colonizes different host niches as part of the normal human microbiota. Proliferation of C. albicans is regulated through a complex balance of host immune defense mechanisms and fungal responses. Expression of AMPs against pathogenic fungi is differentially regulated and initiated by interactions of a variety of fungal pathogen-associated molecular patterns (PAMPs) with pattern recognition receptors (PRRs) on human cells. Inflammatory signaling and other environmental stimuli are also essential to control fungal proliferation and to prevent parasitism. To persist in the host, C. albicans has developed a three-phase AMP evasion strategy, including secretion of peptide effectors, AMP efflux pumps, and regulation of signaling pathways. These mechanisms prevent C. albicans from the antifungal activity of the major AMP classes, including cathelicidins, histatins, and defensins leading to a basal resistance. This minireview summarizes human AMP attack and C. albicans resistance mechanisms and current developments in the use of AMPs as antifungal agents.

Candida albicans cis-prenyltransferase Rer2 is required for protein glycosylation, cell wall integrity and hypha formation.

cis-Prenyltransferase is the first enzyme of the mevalonate pathway committed to the biosynthesis of dolichol in eukaryotes. The RER2 gene encoding cis-prenyltransferase (Rer2p) in the human fungal pathogen Candida albicans was characterized. In addition, the ORF19.5236 encoding the second cis-prenyltransferase, which putatively is responsible for the synthesis of longer polyisoprenoids chains, was identified. When cultivated under repressive conditions, the conditional mutant strain expressing the RER2 gene from the regulatable MET3 promoter contained only 4% of cis-prenyltransferase activity and markedly diminished amounts of dolichols, as compared to the wild-type strain. Moreover, transcriptomal analyses revealed changes in the expression of 300 genes, mainly involved in transport, response to stress, filamentous growth and organelle organization. Growth of the conditional strain was blocked completely at 37 °C. The strain was hypersensitive to a wide range of inhibitors, which suggested glycosylation defects and compromised cell wall integrity. Moreover, the rer2 conditional mutant grown in the repressive conditions, unlike the same strain in the absence of repressor, failed to form hyphae. The results indicate that dolichols are essential not only for protein glycosylation and cell wall integrity but also for growth and development of C. albicans.

Uml2 is a novel CalB-type lipase of Ustilago maydis with phospholipase A activity.

CalB of Pseudozyma aphidis (formerly named Candida antarctica) is one of the most widely applied enzymes in industrial biocatalysis. Here, we describe a protein with 66 % sequence identity to CalB, designated Ustilago maydis lipase 2 (Uml2), which was identified as the product of gene um01422 of the corn smut fungus U. maydis. Sequence analysis of Uml2 revealed the presence of a typical lipase catalytic triad, Ser-His-Asp with Ser125 located in a Thr-Xaa-Ser-Xaa-Gly pentapeptide. Deletion of the uml2 gene in U. maydis diminished the ability of cells to hydrolyse fatty acids from tributyrin or Tween 20/80 substrates, thus demonstrating that Uml2 functions as a lipase that may contribute to nutrition of this fungal pathogen. Uml2 was heterologously produced in Pichia pastoris and recombinant N-glycosylated Uml2 protein was purified from the culture medium. Purified Uml2 released short- and long-chain fatty acids from p-nitrophenyl esters and Tween 20/80 substrates. Furthermore, phosphatidylcholine substrates containing long-chain saturated or unsaturated fatty acids were effectively hydrolysed. Both esterase and phospholipase A activity of Uml2 depended on the Ser125 catalytic residue. These results indicate that Uml2, in contrast to CalB, exhibits not only esterase and lipase activity but also phospholipase A activity. Thus, by genome mining, we identified a novel CalB-like lipase with different substrate specificities.

Morphogenesis-regulated localization of protein kinase A to genomic sites in Candida albicans.

BACKGROUND: The human fungal pathogen Candida albicans is able to undergo morphogenesis from a yeast to a hyphal growth form. Protein kinase A (PKA) isoforms Tpk1 and Tpk2 promote hyphal growth in a signalling pathway via the transcription factor Efg1. RESULTS: C. albicans strains producing epitope-tagged Tpk1 or Tpk2 were used in genome-wide chromatin immunoprecipitation on chip (ChIP chip) to reveal genomic binding sites. During yeast growth, both PKA isoforms were situated primarily within ORFs but moved to promoter regions shortly after hyphal induction. Binding sequences for Tpk2 greatly exceeded Tpk1 sites and did not coincide with binding of the PKA regulatory subunit Bcy1. Consensus binding sequences for Tpk2 within ORFs included ACCAC and CAGCA motifs that appeared to bias codon usage within the binding regions. Promoter residency of Tpk2 correlated with the transcript level of the corresponding gene during hyphal morphogenesis and occurred near Efg1 binding sites, mainly on genes encoding regulators of morphogenesis. CONCLUSIONS: PKA isoforms change their genomic binding sites from ORF to promoter regions during yeast-hyphal morphogenesis. Tpk2 binds preferentially to promoters of genes encoding regulators of cellular morphogenesis.

Candida albicans mucin Msb2 is a broad-range protectant against antimicrobial peptides.

The human fungal pathogen Candida albicans releases a large glycofragment of the Msb2 surface protein (Msb2*) into the growth environment, which protects against the action of human antimicrobial peptides (AMPs) LL-37 and histatin-5. Quantitation of Msb2*/LL-37 interactions by microscale thermophoresis revealed high-affinity binding (dissociation constant [KD] = 73 nM), which was lost or greatly diminished by lack of O-glycosylation or by Msb2* denaturation. Msb2* also interacted with human α- and ß-defensins and protected C. albicans against these AMPs. In addition, the lipopeptide antibiotic daptomycin was bound and inactivated by Msb2*, which prevented the killing of bacterial pathogens Staphylococcus aureus, Enterococcus faecalis, and Corynebacterium pseudodiphtheriticum. In coculturings or mixed biofilms of S. aureus with C. albicans wild-type but not msb2 mutant strains, the protective effects of Msb2* on the bactericidal action of daptomycin were demonstrated. These results suggest that tight binding of shed Msb2* to AMPs that occurs during bacterial coinfections with C. albicans compromises antibacterial therapy by inactivating a relevant reserve antibiotic.