Search

To adapt to various host microenvironments, the human fungal pathogen Candida albicans possesses the capacity to accumulate and store glycogen as an internal carbohydrate source. In the model yeast Saccharomyces cerevisiae , Sc Glc7p and Sc Gac1p are the serine/threonine type 1 protein phosphatase catalytic and regulatory subunits that control glycogen synthesis by altering the phosphorylation state of the glycogen synthase Gsy2p. Despite recent delineation of the glycogen synthesis pathway in C. albicans , the molecular events driving synthase activation are currently undefined. In this study, using a combination of microbiologic and genetic techniques, we determined that the protein encoded by uncharacterized gene C1_01140C , and not the currently annotated C. albicans Gac1p, is the major regulatory subunit involved in glycogen synthesis. C1_01140Cp contains a conserved GVNK motif observed across multiple starch/glycogen-binding proteins in various species, and alanine substitution of each residue in this motif significantly impaired glycogen accumulation in C. albicans . Fluorescent protein tagging and microscopy indicated that C1_01140Cp-GFPy colocalized with Ca Glc7p-tdTomato and Ca Gsy1p-tdTomato accordingly. Co-immunoprecipitation assays further confirmed that C1_01140Cp associates with Ca Glc7p and Ca Gsy1p during glycogen synthesis. Lastly, c1_01140c Δ/Δ exhibited colonization defects in a murine model of vulvovaginal candidiasis. Collectively, our data indicate that uncharacterized C1_01140Cp is the functional ortholog of the PPP1R subunit Sc Gac1p in C. albicans .IMPORTANCEThe capacity to synthesize glycogen offers microbes metabolic flexibility, including the fungal pathogen Candida albicans . In Saccharomyces cerevisiae , dephosphorylation of glycogen synthase by the Sc Glc7p-containing phosphatase is a critical rate-limiting step in glycogen synthesis. Subunits, including Sc Gac1p, target Sc Glc7p to α-1,4-glucosyl primers for efficient Sc Gsy2p synthase activation. However, this process in C. albicans had not been delineated. Here, we show that the C. albicans genome encodes for two homologous phosphatase-binding subunits, annotated Ca Gac1p and uncharacterized C1_01140Cp, both containing a GVNK motif required for polysaccharide affinity. Surprisingly, loss of Ca Gac1p only moderately reduced glycogen accumulation, whereas loss of C1_01140Cp ablated it. Fluorescence microscopy and co-immunoprecipitation approaches revealed that C1_01140Cp associates with Ca Glc7p and Ca Gsy1p during glycogen synthesis. Moreover, C1_01140Cp contributed to fungal fitness at the vaginal mucosa during murine vaginitis. Therefore, this work demonstrates that glycogen synthase regulation is conserved in C. albicans and C1_01140Cp is the functional ortholog of Sc Gac1p., Competing Interests: The authors declare no conflict of interest.

Candidalysin, a cytolytic peptide produced by the fungal pathogen Candida albicans, is a key virulence factor. However, its host cell targets remain elusive. Here we performed a genome-wide loss-of-function CRISPR screen in the TR146 human oral epithelial cell line and identified that disruption of genes (XYLT2, B3GALT6 and B3GAT3) in glycosaminoglycan (GAG) biosynthesis conferred resistance to damage induced by candidalysin and live C. albicans. Surface plasmon resonance and atomic force and electron microscopy indicated that candidalysin binds to sulfated GAGs, facilitating its enrichment on the host cell surface. Adding exogenous sulfated GAGs or the analogue dextran sulfate protected cells against candidalysin-induced damage. Dextran sulfate also inhibited C. albicans invasion and fungal-induced epithelial cell cytokine production. In mice with vulvovaginal candidiasis, topical dextran sulfate administration reduced intravaginal tissue damage and inflammation. Collectively, sulfated GAGs are epithelial cell targets of candidalysin and can be used therapeutically to protect cells from candidalysin-induced damage., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The uridine derivatives UDP-glucose and UDP- N -acetylglucosamine are important for cell wall construction as they are the precursors for the synthesis of β-1,3-glucan and chitin, respectively. Previous studies have demonstrated attenuated virulence of uridine auxotrophs in mice, which has been attributed to insufficient uridine levels for growth in the host. We have discovered that uridine deprivation in the uridine auxotroph ura3 ΔΔ disrupts cell wall architecture by increasing surface mannans, exposing β-1,3-glucan and chitin, and decreasing UDP-sugar levels. Cell wall architecture and UDP-sugars can be rescued with uridine supplementation. The cell wall architectural disruptions in the ura3 ΔΔ mutant also impact immune activation since the mutant elicited greater TNFα secretion from RAW264.7 macrophages than wild type. To determine if cell wall defects contributed to decreased virulence in the ura3 ΔΔ mutant, we used a murine model of systemic infection. Mice infected with the ura3 ΔΔ mutant exhibited increased survival and reduced kidney fungal burden compared with mice infected with wild type. However, suppression of the immune response with cyclophosphamide did not rescue virulence in mice infected with the ura3 ΔΔ mutant, indicating the attenuation in virulence of uridine auxotrophs can be attributed to decreased growth in the host but not increased exposure of β-1,3-glucan. Moreover, the ura3 ΔΔ mutant is unable to grow on ex vivo kidney agar, which demonstrates its inability to colonize the kidneys due to poor growth. Thus, although uridine auxotrophy elicits changes to cell wall architecture that increase the exposure of immunogenic polymers, metabolic fitness costs more strongly drive the observed virulence attenuation.IMPORTANCE Candida albicans is a common cause of bloodstream infections (candidemia). Treatment of these bloodstream infections is made difficult because of increasing antifungal resistance and drug toxicity. Thus, new tactics are needed for antifungal drug development, with immunotherapy being of particular interest. The cell wall of C. albicans is composed of highly immunogenic polymers, particularly β-1,3-glucan. However, β-1,3-glucan is naturally masked by an outer layer of mannoproteins, which hampers the detection of the fungus by the host immune system. Alteration in cell wall components has been shown to increase β-1,3-glucan exposure; however, it is unknown how the inability to synthesize precursors to cell wall components affects unmasking. Here, we demonstrate how cell wall architecture is altered in response to a deficit in precursors for cell wall synthesis and how uridine is a crucial component of these precursors., Competing Interests: The authors declare no conflict of interest.

Enteric pathogens engage in complex interactions with the host and the resident microbiota to establish gut colonization. Although mechanistic interactions between enteric pathogens and bacterial commensals have been extensively studied, whether and how commensal fungi affect pathogenesis of enteric infections remains largely unknown. Here we show that colonization with the common human gut commensal fungus Candida albicans worsened infections with the enteric pathogen Salmonella enterica serovar Typhimurium. Presence of C. albicans in the mouse gut increased Salmonella cecum colonization and systemic dissemination. We investigated the underlying mechanism and found that Salmonella binds to C. albicans via Type 1 fimbriae and uses its Type 3 Secretion System (T3SS) to deliver effector proteins into C. albicans . A specific effector, SopB, was sufficient to manipulate C. albicans metabolism, triggering increased arginine biosynthesis in C. albicans and the release of millimolar amounts of arginine into the extracellular environment. The released arginine, in turn, induced T3SS expression in Salmonella , increasing its invasion of epithelial cells. C. albicans deficient in arginine production was unable to increase Salmonella virulence in vitro or in vivo . In addition to modulating pathogen invasion, arginine also directly influenced the host response to infection. Arginine-producing C. albicans dampened the inflammatory response during Salmonella infection, whereas C. albicans deficient in arginine production did not. Arginine supplementation in the absence of C. albicans increased the systemic spread of Salmonella and decreased the inflammatory response, phenocopying the presence of C. albicans . In summary, we identified C. albicans colonization as a susceptibility factor for disseminated Salmonella infection, and arginine as a central metabolite in the cross-kingdom interaction between fungi, bacteria, and host.

Invasive fungal infections impose an enormous clinical, social, and economic burden on humankind. One of the most common species responsible for invasive fungal infections is Candida albicans . More than 30% of patients with disseminated candidiasis fail therapy with existing antifungal drugs, including the widely used azole class. We previously identified a collection of 13 medications that antagonize the activity of the azoles on C. albicans . Although gain-of-function mutations responsible for antifungal resistance are often associated with reduced fitness and virulence, it is currently unknown how exposure to azole antagonistic drugs impacts C. albicans physiology, fitness, or virulence. In this study, we examined how exposure to seven azole antagonists affects C. albicans phenotype and capacity to cause disease. Most of the azole antagonists appear to have little impact on fungal growth, morphology, stress tolerance, or gene transcription. However, aripiprazole had a modest impact on C. albicans hyphal growth and increased cell wall chitin content. It also aggravated the disseminated C. albicans infections in mice. This effect was abrogated in immunosuppressed mice, indicating that it is at least in part dependent upon host immune responses. Collectively, these data provide proof of principle that unanticipated drug-fungus interactions have the potential to influence the incidence and outcomes of invasive fungal disease., Competing Interests: The authors declare no conflict of interest.

Candida albicans and Staphylococcus aureus are two commonly associated pathogens that cause nosocomial infections with high morbidity and mortality. Our prior and current work using a murine model of polymicrobial intra-abdominal infection (IAI) demonstrates that synergistic lethality is driven by Candida-induced upregulation of functional S. aureus α-toxin leading to polymicrobial sepsis and organ damage. In order to determine the candidal effector(s) mediating enhanced virulence, an unbiased screen of C. albicans transcription factor mutants was undertaken revealing that zcf13Δ/Δ fails to drive augmented α-toxin or lethal synergism during co-infection. A combination of transcriptional and phenotypic profiling approaches shows that ZCF13 regulates genes involved in pentose metabolism, including RBK1 and HGT7 that contribute to fungal ribose catabolism and uptake, respectively. Subsequent experiments reveal that ribose inhibits the staphylococcal agr quorum sensing system and concomitantly represses toxicity. Unlike wild-type C. albicans, zcf13Δ/Δ did not effectively utilize ribose during co-culture or co-infection leading to exogenous ribose accumulation and agr repression. Forced expression of RBK1 and HGT7 in the zcf13Δ/Δ mutant fully restores pathogenicity during co-infection. Collectively, our results detail the interwoven complexities of cross-kingdom interactions and highlight how intermicrobial metabolism impacts polymicrobial disease pathogenesis with devastating consequences for the host., (© 2024. The Author(s).)

Candidalysin is a cytolytic peptide produced by the opportunistic fungal pathogen Candida albicans. This peptide is a key virulence factor in mouse models of mucosal and hematogenously disseminated candidiasis. Despite intense interest in the role of candidalysin in C. albicans pathogenicity, its host cell targets have remained elusive. To fill this knowledge gap, we performed a genome-wide loss-of-function CRISPR screen in a human oral epithelial cell line to identify specific host factors required for susceptibility to candidalysin-induced cellular damage. Among the top hits were XYLT2 , B3GALT6 and B3GAT3 , genes that function in glycosaminoglycan (GAG) biosynthesis. Deletion of these genes led to the absence of GAGs such as heparan sulfate on the epithelial cell surface and increased resistance to damage induced by both candidalysin and live C. albicans. Biophysical analyses including surface plasmon resonance and atomic force and electron microscopy indicated that candidalysin physically binds to sulfated GAGs, facilitating its oligomerization or enrichment on the host cell surface. The addition of exogenous sulfated GAGs or the GAG analogue dextran sulfate protected cells against candidalysin-induced damage. Dextran sulfate, but not non-sulfated dextran, also inhibited epithelial cell endocytosis of C. albicans and fungal-induced epithelial cell cytokine and chemokine production. In a murine model of vulvovaginal candidiasis, topical dextran sulfate administration reduced host tissue damage and decreased intravaginal IL-1β and neutrophil levels. Collectively, these data indicate that GAGs are epithelial cell targets of candidalysin and can be used therapeutically to protect cells from candidalysin-induced damage.

Background: Use of mixed-oil (MO) intravenous fat emulsion (IFE) was shown to inhibit Candida albicans biofilm formation and overall rate of catheter-related bloodstream infections (CR-BSIs) compared with soybean-oil (SO) IFE). We aimed to delineate this inhibitory mechanism and impact of IFE choice on distribution of fungal CR-BSIs., Methods: Transcriptional profiling was conducted on C. albicans grown in SO-IFE, MO-IFE, or SO-IFE with capric acid. Overexpression strains of shared down-regulated genes were constructed using a tetracycline-off system to assess hypha and biofilm formation in IFEs. A 5-year retrospective multicenter cohort study was performed to assess differences in CR-BSIs caused by Candida species based on the IFE formulation received in pediatric patients., Results: Genes significantly down-regulated in MO-IFE and SO-IFE with capric acid included CDC11, HGC1, and UME6. Overexpression of HGC1 or UME6 enabled filamentation in capric acid and MO-IFE. Interestingly, only overexpression of UME6 was sufficient to rescue biofilm growth in MO-IFE. MO-IFE administration was associated with a higher proportion of non-albicans Candida versus C. albicans CR-BSIs (42% vs 33%; odds ratio, 1.22 [95% confidence interval, .46-3.26])., Conclusions: MO-IFE affects C. albicans biofilm formation and hyphal growth via a UME6-dependent mechanism. A numerical but not statistically significant difference in distribution of Candida spp. among CR-BSIs was observed., Competing Interests: Potential conflicts of interest . All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

While the fungus Candida albicans is a common colonizer of healthy humans, it is also responsible for mucosal infections and severe invasive disease. Understanding the mechanisms that allow C. albicans to exist as both a benign commensal and as an invasive pathogen have been the focus of numerous studies, and recent findings indicate an important role for cross-kingdom interactions on C. albicans biology. This review highlights how C. albicans-bacteria interactions influence healthy polymicrobial community structure, host immune responses, microbial pathogenesis, and how dysbiosis may lead to C. albicans infection. Finally, we discuss how cross-kingdom interactions represent an opportunity to identify new antivirulence compounds that target fungal infections., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The mammalian gut secretes a family of multifunctional peptides that affect appetite, intestinal secretions, and motility whereas others regulate the microbiota. We have found that peptide YY (PYY 1-36 ), but not endocrine PYY 3-36 , acts as an antimicrobial peptide (AMP) expressed by gut epithelial paneth cells (PC). PC-PYY is packaged into secretory granules and is secreted into and retained by surface mucus, which optimizes PC-PYY activity. Although PC-PYY shows some antibacterial activity, it displays selective antifungal activity against virulent Candida albicans hyphae-but not the yeast form. PC-PYY is a cationic molecule that interacts with the anionic surfaces of fungal hyphae to cause membrane disruption and transcriptional reprogramming that selects for the yeast phenotype. Hence, PC-PYY is an antifungal AMP that contributes to the maintenance of gut fungal commensalism.

The polymorphic fungus Candida albicans remains a leading cause of both invasive and superficial mycoses, including vulvovaginal candidiasis (VVC). Metabolic plasticity, including carbohydrate catabolism, confers fitness advantages at anatomical site-specific host niches. C. albicans possesses the capacity to accumulate and store carbohydrates as glycogen and can consume intracellular glycogen stores when nutrients become limited. In the vaginal environment, estrogen promotes epithelial glycogen accumulation and C. albicans colonization. However, whether these factors are mechanistically linked is unexplored. Here, we characterized the glycogen metabolism pathways in C. albicans and investigated whether these impact the long-term survival of C. albicans, both in vitro and in vivo during murine VVC, or virulence during systemic infection. SC5314 and 6 clinical isolates demonstrated impaired growth when glycogen was used as the sole carbon source, suggesting that environmental glycogen acquisition is limited. The genetic deletion and complementation of key genes involved in glycogen metabolism in Saccharomyces cerevisiae confirmed that GSY1 and GLC3 , as well as GPH1 and GDB1 , are essential for glycogen synthesis and catabolism in C. albicans, respectively. Potential compensatory roles for a glucoamylase encoded by SGA1 were also explored. Competitive survival assays revealed that gsy1Δ/Δ , gph1 Δ/Δ, and gph1Δ/Δ sga1Δ/Δ mutants exhibited long-term survival defects in vitro under starvation conditions and in vivo during vaginal colonization. A complete inability to catabolize glycogen ( gph1Δ/Δ sga1Δ/Δ ) also rendered C. albicans significantly less virulent during disseminated infections. This is the first study fully validating the glycogen metabolism pathways in C. albicans, and the results further suggest that intracellular glycogen catabolism positively impacts the long-term fitness of C. albicans in nutrient deficient environments and is important for full virulence. IMPORTANCE Glycogen is a highly branched polymer of glucose and is used across the tree of life as an efficient and compact form of energy storage. Whereas glycogen metabolism pathways have been studied in model yeasts, they have not been extensively explored in pathogenic fungi. Using a combination of microbiologic, molecular genetic, and biochemical approaches, we reveal orthologous functions of glycogen metabolism genes in the fungal pathogen Candida albicans. We also provide evidence that extracellular glycogen poorly supports growth across the Candida species and clinical isolates. Competitive fitness assays reveal that the loss of glycogen synthesis or catabolism significantly impacts survival during both in vitro starvation and the colonization of the mouse vagina. Moreover, a global glycogen catabolism mutant is rendered less virulent during murine invasive candidiasis. Therefore, this work demonstrates that glycogen metabolism in C. albicans contributes to survival and virulence in the mammalian host and may be a novel antifungal target.

Candida albicans is a deadly pathogen responsible for millions of mucosal and systemic infections per year. The pathobiology of C. albicans is largely dependent on the damaging and immunostimulatory properties of the peptide candidalysin (CL), a key virulence factor. When CL forms pores in the plasma membrane of epithelial cells, it activates a response network grounded in activation of the epidermal growth factor receptor. Prior reviews have characterized the resulting CL immune activation schemas but lacked insights into the molecular mechanism of CL membrane damage. We recently demonstrated that CL functions by undergoing a unique self-assembly process; CL forms polymers and loops in aqueous solution prior to inserting and forming pores in cell membranes. This mechanism, the first of its kind to be observed, informs new therapeutic avenues to treat Candida infections. Recently, variants of CL were identified in other Candida species, providing an opportunity to identify the residues that are key for CL to function. In this review, we connect the ability of CL to damage cell membranes to its immunostimulatory properties., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Shielding the immunogenic cell wall epitope β(1, 3)-glucan under an outer layer of mannosylated glycoproteins is an essential virulence factor deployed by Candida albicans during systemic infection. Accordingly, mutants with increased β(1, 3)-glucan exposure (unmasking) display increased immunostimulatory capabilities in vitro and attenuated virulence during systemic infection in mice. However, little work has been done to assess the impact of increased unmasking during the two most common manifestations of candidiasis, namely, oropharyngeal candidiasis (OPC) and vulvovaginal candidiasis (VVC). We have shown previously that the expression of a single hyperactive allele of the MAP3K STE11 ΔN467 induces unmasking via the Cek1 MAPK pathway, attenuates fungal burden, and prolongs survival during systemic infection in mice. Here, we expand on these findings and show that infection with an unmasked STE11 ΔN467 mutant also impacts disease progression during OPC and VVC murine infection models. Male mice sublingually infected with the STE11 ΔN467 mutant showed a significant reduction in tongue fungal burden at 2 days postinfection and a modest reduction at 5 days postinfection. However, we find that selection for STE11 ΔN467 suppressor mutants that no longer display increased unmasking occurs within the oral cavity and is likely responsible for the restoration of fungal burden trends to wild-type levels later in the infection. In the VVC infection model, no attenuation in fungal burden was observed. However, polymorphonuclear cell recruitment and interleukin-1β (IL-1β) levels within the vaginal lumen, markers of immunopathogenesis, were increased in mice infected with unmasked STE11 ΔN467 cells. Thus, our data suggest a niche-specific impact for unmasking on disease progression.

Study Objective: To compare rates of catheter-related bloodstream infections (CR-BSI) in pediatric patients who received parenteral nutrition (PN) with either soybean oil-based intravenous fat emulsion (SO-IFE) or mixed oil-IFE (MO-IFE). We hypothesized that the use of MO-IFE would be independently associated with reduced infection rates compared with SO-IFE., Design: Retrospective cohort study., Setting: Tertiary referral children's hospital and its associated gastrointestinal rehabilitation clinic (01 January, 2015-31 July, 2019)., Patients: Days of IFE exposure were counted for patients aged <18 years on IFE initiated during the review period, who had a central venous catheter (CVC) placed for PN administration, received IFE at least three times weekly, and for at least 7 days., Measurements: The primary outcome included total and categorical CR-BSI rates expressed as the average with standard error (SE) number of infections per 1000 fat emulsion days. The following categories were specified: Candida albicans, non-albicans Candida spp., coagulase-negative Staphylococcus (CoNS), Enterobacterales, methicillin-resistant S. aureus, methicillin-susceptible S. aureus, and Pseudomonadales. Average infection rate comparisons were quantified as incidence rate ratios (IRR) using generalized linear mixed modeling with a Poisson distribution., Main Results: Seven hundred and forty-three SO-IFE and 450 MO-IFE exposures were reviewed from 1131 patients, totaling 37,599 and 19,796 days of therapy, respectively. From those found significantly different, the average rate of infections with CoNS was 3.58 (SE 0.5)/1000 days of SO-IFE and 1.39 (SE 0.45)/1000 days of MO-IFE (IRR [95% confidence interval, CI]: 0.27 [0.16-0.46]; p < 0.01). Total average rates of infection were 7.33 (SE 0.76)/1000 days of SO-IFE and 4.52 (SE 0.75)/1000 days of MO-IFE (IRR [95% CI]: 0.60 [0.44-0.81]; p < 0.01). Other factors associated with higher infection rates include female gender, neonatal age, and inpatient-only IFE exposure., Conclusions: Receipt of MO-IFE was associated with lower rates of CoNS and total CR-BSIs compared with SO-IFE in pediatric patients. These findings could have major implications on IFE selection for pediatric patients receiving PN., (© 2022 Pharmacotherapy Publications, Inc.)

As increasing evidence emerges that interstrain genetic diversity among Candida albicans clinical isolates underpins phenotypic variation compared to the reference isolate SC5314, new genetic tools are required to interrogate gene function across strain backgrounds. Here, the SAT1 -flipper plasmid was reengineered to contain a C. albicans codon optimized hygromycin B resistance gene ( CaHygB ). Cassettes were PCR-amplified from both SAT1 -flipper and CaHygB -flipper plasmids using primers with homologous sequences flanking target genes of interest to serve as repair templates. Ribonucleoprotein (RNP) complexes containing proprietary CRISPR RNAs (crRNAs), universal transactivating CRISPR RNA (tracrRNA), and Cas9 protein were assembled in vitro and transformed, along with both repair templates, by electroporation into C. albicans. Homozygous deletion of the ADE2 gene results in red-pigmented colonies and this gene was used to validate our approach. Both in SC5314 and a variety of clinical isolates (529L, JS15, SJCA1, TW1), homozygous gene targeting was nearly 100% when plating on media containing nourseothricin and hygromycin B with transformation efficiencies exceeding 10 4 homozygous deletion mutants per μg of DNA. A gene reversion system was also employed with plasmids pDUP3 and pDIS3 engineered to contain the ADH1 terminator and an overlap extension PCR-mediated approach combined with CRISPR-Cas9 targeting at the NEUT5 neutral locus. A variety of single or compound mutants (Δ/Δ als3 , Δ/Δ cph1 Δ/Δ efg1 , Δ/Δ ece1 ) and their revertant strains were constructed and phenotypically validated by a variety of assays, including biofilm formation, hyphal growth, and macrophage IL-1β response. Thus, we have established a cloning-free, modular system for highly efficient homozygous gene deletion and reversion in diverse isolates. IMPORTANCE Recently, phenotypic heterogeneity in Candida albicans isolates has been recognized as an underappreciated factor contributing to gene diversification and broadly impacts strain-to-strain antifungal resistance, fitness, and pathogenicity. We have designed a cloning-free genetic system for rapid gene deletion and reversion in C. albicans clinical isolates that interlaces established recyclable genetic systems with CRISPR-Cas9 technology. The SAT1 -flipper was reengineered to contain CaHygB encoding resistance to hygromycin B. Using a modular PCR-mediated approach coupled with in vitro ribonucleoprotein assembly with commercial reagents, both SAT1 - and CaHygB -flipper cassettes were simultaneously integrated at loci with high efficiency (10 4 transformants per μg DNA) and upward of 99% homozygous gene targeting across a collection of diverse isolates of various anatomical origin. Revertant strains were constructed by overlap extension PCR with CRISPR-Cas9 targeted integration at the NEUT5 locus. Thus, this facile system will aid in unraveling the genetic factors contributing to the complexity of intraspecies diversity.

Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving immunopathology at the vaginal mucosa. This study aimed to determine if expression and/or processing of Ece1p differs across C. albicans isolates and whether this partly underlies differential pathogenicity observed clinically. Using a targeted sequencing approach, we determined that isolate 529L harbors a similarly expressed, yet distinct Ece1p isoform variant that encodes for a predicted functional candidalysin; this isoform was conserved amongst a collection of clinical isolates. Expression of the ECE1 open reading frame (ORF) from 529L in an SC5314-derived ece1Δ/Δ strain resulted in significantly reduced vaginopathogenicity as compared to an isogenic control expressing a wild-type (WT) ECE1 allele. However, in vitro challenge of vaginal epithelial cells with synthetic candidalysin demonstrated similar toxigenic activity amongst SC5314 and 529L isoforms. Creation of an isogenic panel of chimeric strains harboring swapped Ece1p peptides or HiBiT tags revealed reduced secretion with the ORF from 529L that was associated with reduced virulence. A genetic survey of 78 clinical isolates demonstrated a conserved pattern between Ece1p P2 and P3 sequences, suggesting that substrate specificity around Kex2p-mediated KR cleavage sites involved in protein processing may contribute to differential pathogenicity amongst clinical isolates. Therefore, we present a new mechanism for attenuation of C. albicans virulence at the ECE1 locus., Competing Interests: The authors have declared that no competing interests exist.

Invasive and superficial infections caused by the Candida species result in significant global morbidity and mortality. As the pathogenicity of these organisms is intimately intertwined with host immune response, therapies to target both the fungus and host inflammation may be warranted. Structural similarities exist between established inhibitors of the NLRP3 inflammasome and those of fungal acetohydroxyacid synthase (AHAS). Therefore, we leveraged this information to conduct an in silico molecular docking screen to find novel polypharmacologic inhibitors of these targets that resulted in the identification of 12 candidate molecules. Of these, compound 10 significantly attenuated activation of the NLPR3 inflammasome by LPS + ATP, while also demonstrating growth inhibitory activity against C. albicans that was alleviated in the presence of exogenous branched chain amino acids, consistent with targeting of fungal AHAS. SAR studies delineated an essential molecular scaffold required for dual activity. Ultimately, 10 and its analog 10a resulted in IC 50 (IL-1β release) and MIC 50 (fungal growth) values with low μM potency against several Candida species. Collectively, this work demonstrates promising potential of dual-target approaches for improved management of fungal infections.

Although considered effective treatment for many yeast fungi, the therapeutic efficacy of the echinocandin class of antifungals for invasive aspergillosis (IA) is limited. Recent studies suggest intense kinase- and phosphatase-mediated echinocandin adaptation in A. fumigatus. To identify A. fumigatus protein kinases required for survival under echinocandin stress, we employed CRISPR/Cas9-mediated gene targeting to generate a protein kinase disruption mutant library in a wild type genetic background. Cell wall and echinocandin stress screening of the 118 disruption mutants comprising the library identified only five protein kinase disruption mutants displaying greater than 4-fold decreased echinocandin minimum effective concentrations (MEC) compared to the parental strain. Two of these mutated genes, the previously uncharacterized A. fumigatus sepL and sidB genes, were predicted to encode protein kinases functioning as core components of the Septation Initiation Network (SIN), a tripartite kinase cascade that is necessary for septation in fungi. As the A. fumigatus SIN is completely uncharacterized, we sought to explore these network components as effectors of echinocandin stress survival. Our data show that mutation of any single SIN kinase gene caused complete loss of hyphal septation and increased susceptibility to cell wall stress, as well as widespread hyphal damage and loss of viability in response to echinocandin stress. Strikingly, mutation of each SIN kinase gene also resulted in a profound loss of virulence characterized by lack of tissue invasive growth. Through the deletion of multiple novel regulators of hyphal septation, we show that the non-invasive growth phenotype is not SIN-kinase dependent, but likely due to hyphal septation deficiency. Finally, we also find that echinocandin therapy is highly effective at eliminating residual tissue burden in mice infected with an aseptate strain of A. fumigatus. Together, our findings suggest that inhibitors of septation could enhance echinocandin-mediated killing while simultaneously limiting the invasive potential of A. fumigatus hyphae., Competing Interests: The authors have declared that no competing interests exist.

The coordination of single or multiple microorganisms are required for the manufacture of traditional fermented foods, improving the flavour and nutrition of the food materials. However, both the additional economic benefits and safety concerns have been raised by microbiotas in fermented products. Among the fermented products, Lactobacillus and Saccharomyces cerevisiae are one of the stable microbiotas, suggesting their interaction is mediated by coexistence-relevant mechanisms and prevent to be excluded by other microbial species. Thus, aiming to guide the manufacture of fermented foods, this review will focus on interactions of coexistence-relevant mechanisms between Lactobacillus and S. cerevisiae , including metabolites communications, aggregation, and polymicrobial biofilm. Also, the molecular regulatory network of the coexistence-relevant mechanisms is discussed according to omics researches.

While human vaginal pH in childbearing-age women is conclusively acidic, the mouse vaginal pH is reported as being near neutral. However, this information appears to be somewhat anecdotal with respect to vulvovaginal candidiasis, as such claims in the literature frequently lack citations of studies that specifically address this physiological factor. Given the disparate pH between mice and humans, the role of exogenous hormones and colonization by the fungal pathogen Candida albicans in shaping vaginal pH was assessed. Use of a convenient modified vaginal lavage technique with the pH indicator dye phenol red demonstrated that indeed vaginal pH was near neutral (7.2 ± 0.24) and was not altered by delivery of progesterone or estrogen in C57BL/6 mice. These trends were conserved in DBA/2 and CD-1 mouse backgrounds, commonly used in the mouse model of vaginitis. It was also determined that vaginal colonization with C. albicans did not alter the globally neutral vaginal pH over the course of one week. Construction and validation of a C. albicans reporter strain expressing GFPy, driven by the pH-responsive PHR1 promoter, confirmed the murine vaginal pH to be at least ≥6.0. Collectively, our data convincingly demonstrate a stable and conserved near neutrality of the mouse vaginal pH during vulvovaginal candidiasis and should serve as a definitive source for future reference. Implications and rationale for disparate pH in this model system are also discussed., (Copyright © 2021 American Society for Microbiology.)

Candida albicans, a ubiquitous commensal fungus that colonizes human mucosal tissues and skin, can become pathogenic, clinically manifesting most commonly as oropharyngeal candidiasis and vulvovaginal candidiasis (VVC). Studies in mice and humans convincingly show that T-helper 17 (Th17)/interleukin 17 (IL-17)-driven immunity is essential to control oral and dermal candidiasis. However, the role of the IL-17 pathway during VVC remains controversial, with conflicting reports from human data and mouse models. Like others, we observed induction of a strong IL-17-related gene signature in the vagina during estrogen-dependent murine VVC. As estrogen increases susceptibility to vaginal colonization and resulting immunopathology, we asked whether estrogen use in the standard VVC model masks a role for the Th17/IL-17 axis. We demonstrate that mice lacking IL-17RA, Act1, or interleukin 22 showed no evidence for altered VVC susceptibility or immunopathology, regardless of estrogen administration. Hence, these data support the emerging consensus that Th17/IL-17 axis signaling is dispensable for the immunopathogenesis of VVC., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Candida albicans , along with other closely related Candida species, are the primary causative agents of vulvovaginal candidiasis (VVC)-a multifactorial infectious disease of the lower female reproductive tract resulting in pathologic inflammation. Unlike other forms of candidiasis, VVC is a disease of immunocompetent and otherwise healthy women, most predominant during their child-bearing years. While VVC is non-lethal, its high global incidence and profound negative impact on quality-of-life necessitates further understanding of the host and fungal factors that drive disease pathogenesis. In this review, we cover the current state of our understanding of the epidemiology, host response, fungal pathogenicity mechanisms, impact of the microbiome, and novel approaches to treatment of this most prevalent human candidal infection. We also offer insight into the latest advancements in the VVC field and identify important questions that still remain., Competing Interests: The authors declare no conflict of interest.

Repurposing of currently approved medications is an attractive option for the development of novel treatment strategies against physiological and infectious diseases. The antidiabetic sulfonylurea glyburide has demonstrated off-target capacity to inhibit activation of the NLRP3 inflammasome in a variety of disease models, including vaginal candidiasis, caused primarily by the fungal pathogen Candida albicans Therefore, we sought to determine which of the currently approved sulfonylurea drugs prevent the release of interleukin 1β (IL-1β), a major inflammasome effector, during C. albicans challenge of the human macrophage-like THP1 cell line. Findings revealed that the second-generation antidiabetics (glyburide, glisoxepide, gliquidone, and glimepiride), which exhibit greater antidiabetic efficacy than prior iterations, demonstrated anti-inflammatory effects with various degrees of potency as determined by calculation of 50% inhibitory concentrations (IC 50 s). These same compounds were also effective in reducing IL-1β release during noninfectious inflammasome activation (e.g., induced by lipopolysaccharide [LPS] plus ATP), suggesting that their anti-inflammatory activity is not specific to C. albicans challenge. Moreover, treatment with sulfonylurea drugs did not impact C. albicans growth and filamentation or THP1 viability. Finally, the use of ECE1 and Candidalysin deletion mutants, along with isogenic NLRP3 -/- cells, demonstrated that both Candidalysin and NLRP3 are required for IL-1β secretion, further confirming that sulfonylureas suppress inflammasome signaling. Moreover, challenge of THP1 cells with synthetic Candidalysin peptide demonstrated that this toxin is sufficient to activate the inflammasome. Treatment with the experimental inflammasome inhibitor MCC950 led to similar blockade of IL-1β release, suggesting that Candidalysin-mediated inflammasome activation can be inhibited independently of potassium efflux. Together, these results demonstrate that the second-generation antidiabetic sulfonylureas retain anti-inflammatory activity and may be considered for repurposing against immunopathological diseases, including vaginal candidiasis., (Copyright © 2020 American Society for Microbiology.)

Candida albicans and Staphylococcus aureus are common causes of nosocomial infections with severe morbidity and mortality. Murine polymicrobial intra-abdominal infection (IAI) with C. albicans and S. aureus results in acute mortality dependent on the secreted cytolytic effector alpha-toxin. Here, we confirmed that alpha-toxin is elevated during polymicrobial growth compared to monomicrobial growth in vitro Therefore, this study sought to unravel the mechanism by which C. albicans drives enhanced staphylococcal alpha-toxin production. Using a combination of functional and genetic approaches, we determined that an intact agr quorum sensing regulon is necessary for enhanced alpha-toxin production during coculture and that a secreted candidal factor likely is not implicated in elevating agr activation. As the agr system is pH sensitive, we observed that C. albicans raises the pH during polymicrobial growth and that this correlates with increased agr activity and alpha-toxin production. Modulation of the pH could predictably attenuate or activate agr activity during coculture. By using a C. albicans mutant deficient in alkalinization ( stp2 Δ/Δ), we confirmed that modulation of the extracellular pH by C. albicans can drive agr expression and toxin production. Additionally, the use of various Candida species ( C. glabrata , C. dubliniensis , C. tropicalis , C. parapsilosis , and C. krusei ) demonstrated that those capable of raising the extracellular pH correlated with elevated agr activity and alpha-toxin production during coculture. Overall, we demonstrate that alkalinization of the extracellular pH by the Candida species leads to sustained activation of the staphylococcal agr system. IMPORTANCE Candida albicans and Staphylococcus aureus are commonly coisolated from central venous catheters and deep-seated infections, including intra-abdominal sepsis. Thus, they represent a significant cause of nosocomial morbidity and mortality. Yet how these organisms behave in the context of polymicrobial growth remains poorly understood. In this work, we set out to determine the mechanism by which activation of the staphylococcal agr quorum sensing system and production of its major virulence effector alpha-toxin is enhanced during coculture with C. albicans Surprisingly, we likely ruled out that a secreted candidal factor drives this process. Instead, we demonstrated that alkalinization of the extracellular milieu by C. albicans and other Candida species correlated with elevated agr activity. Thus, we propose a mechanism where modulation of the extracellular pH by fungal opportunists can indirectly alter virulence of a bacterial pathogen. Uncovering molecular events that drive interkingdom pathogenicity mechanisms may enhance surveillance and treatment for devastating polymicrobial infections., (Copyright © 2019 Todd et al.)

Fungal and bacterial commensal organisms play a complex role in the health of the human host. Expansion of commensal ecology after birth is a critical period in human immune development. However, the initial fungal colonization of the primordial gut remains undescribed. To investigate primordial fungal ecology, we performed amplicon sequencing and culture-based techniques of first-pass meconium, which forms in the intestine prior to birth, from a prospective observational cohort of term and preterm newborns. Here, we describe fungal ecologies in the primordial gut that develop complexity with advancing gestational age at birth. Our findings suggest homeostasis of fungal commensals may represent an important aspect of human biology present even before birth. Unlike bacterial communities that gradually develop complexity, the domination of the fungal communities of some preterm infants by Saccromycetes, specifically Candida , may suggest a pathologic association with preterm birth.-Willis, K. A., Purvis, J. H., Myers, E. D., Aziz, M. M., Karabayir, I., Gomes, C. K., Peters, B. M., Akbilgic, O., Talati, A. J., Pierre, J. F. Fungi form interkingdom microbial communities in the primordial human gut that develop with gestational age.

Receipt of parenteral nutrition (PN) remains an independent risk factor for developing catheter-related bloodstream infections (CR-BSI) caused by fungi, including by the polymorphic fungus Candida albicans , which is notoriously adept at forming drug-resistant biofilm structures. Among a variety of macronutrients, PN solutions contain lipid emulsions to supply daily essential fats and are often delivered via central venous catheters (CVCs). Therefore, using an in vitro biofilm model system, we sought to determine whether various clinical lipid emulsions differentially impacted biofilm growth in C. albicans We observed that the lipid emulsions Intralipid and Omegaven both stimulated C. albicans biofilm formation during growth in minimal medium or a macronutrient PN solution. Conversely, Smoflipid inhibited C. albicans biofilm formation by approximately 50%. Follow-up studies revealed that while Smoflipid did not impair C. albicans growth, it did significantly inhibit hypha formation and hyphal elongation. Moreover, growth inhibition could be recapitulated in Intralipid when supplemented with capric acid-a fatty acid present in Smoflipid but absent in Intralipid. Capric acid was also found to dose dependently inhibit C. albicans biofilm formation in PN solutions. This is the first study to directly compare different clinical lipid emulsions for their capacity to affect C. albicans biofilm growth. Results derived from this study necessitate further research regarding different lipid emulsions and rates of fungus-associated CR-BSIs., (Copyright © 2019 American Society for Microbiology.)

While Koch's Postulates have established rules for microbial pathogenesis that have been extremely beneficial for monomicrobial infections, new studies regarding polymicrobial pathogenesis defy these standards. The explosion of phylogenetic sequence data has revolutionized concepts of microbial interactions on and within the host. However, there remains a paucity of functional follow-up studies to delineate mechanisms driven by such interactions and how they shape health or disease. That said, one particular microbial pairing, the fungal opportunist Candida albicans and the bacterial pathogen Staphylococcus aureus , has received much attention over the last decade. Therefore, the objective of this review is to discuss the multi-faceted mechanisms employed by these two ubiquitous human pathogens during polymicrobial growth, including how they: establish and persist in inter-Kingdom biofilms, tolerate antimicrobial therapy, co-invade host tissue, exacerbate quorum sensing and staphylococcal toxin production, and elicit infectious synergism. Commentary regarding new challenges and remaining questions related to future discovery of this fascinating fungal-bacterial interaction is also provided.

Candida albicans and Staphylococcus aureus are among the most prevalent nosocomial pathogens that are responsible for severe morbidity and mortality, even with appropriate treatment. Using a murine model of polymicrobial intra-abdominal infection (IAI), we have previously shown that coinfection with these pathogens results in synergistic lethality that is partially dependent on exacerbated prostaglandin signaling, while monomicrobial infection is nonlethal. Therefore, the objective of this study was to identify staphylococcal virulence determinants that drive lethal synergism during polymicrobial IAI. Using the toxigenic S. aureus strain JE2, we observed that coinfection with C. albicans led to a striking 80 to 100% mortality rate within 20 h postinoculation (p.i.) while monomicrobial infections were nonlethal. Use of a green fluorescent protein (GFP)-P3 promoter S. aureus reporter strain revealed enhanced activation of the staphylococcal agr quorum sensing system during in vitro polymicrobial versus monomicrobial growth. Analyses by quantitative real-time PCR (qPCR), Western blot, and toxin functional assays confirmed enhanced agr -associated gene transcription and increases in secreted alpha- and delta-toxins. C. albicans -mediated elevated toxin production and hemolytic activity were determined to be agrA dependent, and genetic knockout and complementation of hla identified alpha-toxin as the key staphylococcal virulence factor driving lethal synergism. Analysis of mono- and polymicrobial infections 8 h p.i. demonstrated equivalent bacterial burdens in the peritoneal cavity but significantly elevated levels of alpha-toxin (3-fold) and the eicosanoid prostaglandin E 2 (PGE 2 ) (4-fold) during coinfection. Importantly, prophylactic passive immunization using the monoclonal anti-alpha-toxin antibody MEDI4893* led to significantly improved survival rates compared to those following treatment with isotype control antibody. Collectively, these results define alpha-toxin as an essential virulence determinant during C. albicans - S. aureus IAI and describe a novel mechanism by which a human-pathogenic fungus can augment the virulence of a highly pathogenic bacterium in vivo IMPORTANCE Relatively little is known about the complex interactions and signaling events that occur between microbes and even less so about how microbial "cross talk" shapes human health and disease. Candida albicans (a fungus) and Staphylococcus aureus (a bacterium) are formidable human nosocomial pathogens, causing severe morbidity and mortality. Moreover, they are frequently coisolated from central venous catheters and deep-seated infections, including intra-abdominal sepsis. In this work, we have shown that coinfection with C. albicans and S. aureus is highly lethal, leading to >80% mortality by day 1 postinfection, whereas monoinfection with C. albicans or S. aureus does not cause mortality. This infectious synergism is dependent on the expression of staphylococcal alpha-toxin, and secretion of this potent virulence factor is actually augmented by C. albicans via an agr -dependent mechanism. Moreover, prophylactic neutralization of alpha-toxin with a monoclonal antibody is sufficient to elicit protection during coinfection. Therefore, we have demonstrated that a pathogenic fungus can enhance virulence determinants of a bacterium in vivo with devastating consequences to the host. These results have important implications in the surveillance and treatment of polymicrobial disease and highlight the dynamic intersection of environment, pathogens, and host., (Copyright © 2019 Todd et al.)

This study aimed to evaluate the Staphylococcus aureus biofilm formation and Nε-carboxymethyl-lysine generation ability under food heat processing conditions including pH (5.0-9.0), temperature (25 °C, 31 °C, 37 °C, 42 °C and 65 °C), NaCl concentration (10%, 15% and 20%, w/v) and glucose concentration (0.5%, 1%, 2%, 3%, 5%, 10%, w/v). S. aureus biofilm genetic character was obtained by PCR detecting atl, ica operon, sasG and agr. Biofilm biomass and metabolic activity were quantified with crystal violet and methyl thiazolyl tetrazolium staining methods. S. aureus biofilm was sensitive to food heat processing conditions with 37 °C, pH 7.0, 2% glucose concentration (w/v) and 10% NaCl concentration (w/v) were favorable conditions. Besides, free and bound Nε-carboxymethyl-lysine level in weak, moderate and strong biofilm were detected by optimized high performance liquid chromatography tandem mass spectrometry. Nε-carboxymethyl-lysine level in S. aureus biofilm possessed a significant gap between strong, moderate and weak biofilm strains. This investigation revealed the biological and chemical hazard of Staphylococcus aureus biofilm to food processing environment.

This study aimed at identification and characterization of a novel multidrug-resistant Pseudomonas putida strain Guangzhou-Ppu420 carrying two copies of qnrVC6 isolated from a hospital in Guangzhou, China, in 2012. Antimicrobial susceptibility was tested by Vitek2™ Automated Susceptibility System and Etest™ strips, and whole-genome sequencing facilitated analysis of its multidrug resistance. The genome has a length of 6,031,212 bp and an average G + C content of 62.01%. A total of 5,421 open reading frames were identified, including eight 5S rRNA, seven 16S rRNA, and seven 23S rRNA, and 76 tRNA genes. Importantly, two copies of qnrVC6 gene with three ISCR1 around, a bla VIM-2 carrying integron In528, a novel gcu173 carrying integron In1348, and six antibiotic resistance genes were identified. This is the first identification of two copies of the qnrVC6 gene in a single P. putida isolate and a class 1 integron In1348.

Background: Fluoroquinolone-resistant Haemophilus influenzae (FRHI) has been reported worldwide but remain unclear in China., Methods: A total of 402 H. influenzae isolates collected from 2016 to 2017 were included. Antimicrobial susceptibility on 10 antibiotics was performed, and minimum inhibitory concentration of ciprofloxacin- and nalidixic acid-resistant strains were further determined by E-test strips, with risk factors also evaluated. Strains with resistance or reduced susceptibility to ciprofloxacin were subjected to sequencing of the quinolone resistance-determining regions (QRDR) and plasmid-mediated quinolone resistance genes by sequencing, with multi-locus sequence typing., Results: 2.2% of H. influenzae strains were non-susceptible (7/402, 1.7%) or susceptible (2/402, 0.5%) to ciprofloxacin but NAL-resistant by E-test, and multidrug resistance was more common in fluoroquinolones non-susceptible H. influenzae group (p = 0.000). Infection risk factors included invasive procedure (p = 0.011), catching cold/previous contact with someone who had a cold (p = 0.019), fluoroquinolones use during previous 3 months (p = 0.003). With none of mutations obtained in gyrB, parE and other plasmid-mediated quinolone resistance genes, 7 and 4 strains were found for Ser-84-Leu substitutions in gyrA and one amino acid substitution in the QRDR of gyrA linked with one amino acid substitution in the QRDR of parC, respectively. In addition, five sequence types (ST) were identified, with ST1719 firstly found., Conclusions: For the first time, this study has reported the incidence, risk factors, molecular determinants on fluoroquinolones resistance and ST of FRHI strains in mainland China, representing the first evidence of mutation of gyrA and parC in China and the new ST1719 worldwide.

The human fungal pathogen Candida albicans is the major etiological agent of vulvovaginal candidiasis (VVC). Despite this fact, other non- albicans Candida (NAC) species have frequently been reported, as well. Despite their presence in the vaginal environment, little is known about their capacities to elicit immune responses classically associated with C. albicans -mediated immunopathology, including neutrophil recruitment and proinflammatory cytokine signaling. Therefore, using a combination of in vitro and in vivo approaches, we undertook a comparative analysis to determine whether a representative panel of NAC species could colonize, induce immunopathological markers, or cause damage at the vaginal mucosa. Using a murine model of VVC, C. albicans was found to induce robust immunopathology (neutrophils and interleukin 1β [IL-1β]) and elicit mucosal damage. However, all the NAC species tested (including C. dubliniensis , C. tropicalis , C. parapsilosis , C. krusei , C. glabrata , and C. auris ) induced significantly less damage and neutrophil recruitment than C. albicans , despite achieving similar early colonization levels. These results largely correlated with a notable lack of ability by the NAC species (including C. dubliniensis and C. tropicalis ) to form hyphae both in vitro and in vivo Furthermore, both C. dubliniensis and C. tropicalis induced significantly less expression of the ECE1 gene encoding candidalysin, a key fungal virulence determinant driving VVC immunopathology. In order to determine the relative capacities of these species to elicit inflammasome-dependent IL-1β release, both wild-type and NLRP3 -/- THP-1 cells were challenged in vitro While most species tested elicited only modest amounts of IL-1β, challenge with C. albicans led to significantly elevated levels that were largely NLRP3 dependent. Collectively, our findings demonstrate that although NAC species are increasingly reported as causative agents of VVC, C. albicans appears to be exceedingly vaginopathogenic, exhibiting robust immunopathology, hypha formation, and candidalysin expression. Thus, this study provides mechanistic insight into why C. albicans is overwhelmingly the major pathogen reported during VVC., (Copyright © 2018 American Society for Microbiology.)

Staphylococcus aureus is one of the representative foodborne pathogens which forms biofilm. Antibiotics are widely applied in livestock husbandry to maintain animal health and productivity, thus contribute to the dissemination of antimicrobial resistant livestock and human pathogens, and pose a significant public health threat. Effect of antibiotic pressure on S. aureus biofilm formation, as well as the mechanism, remains unclear. In this study, the regulatory mechanism of low concentration of ampicillin on S. aureus biofilm formation was elucidated. The viability and biomass of biofilm with and without 1/4 MIC ampicillin treatment for 8 h were determined by XTT and crystal violet straining assays, respectively. Transcriptomics analysis on ampicillin-induced and non-ampicillin-induced biofilms were performed by RNA-sequencing, differentially expressed genes identification and annotation, GO functional and KEGG pathway enrichment. The viability and biomass of ampicillin-induced biofilm showed dramatical increase compared to the non-ampicillin-induced biofilm. A total of 530 differentially expressed genes (DEGs) with 167 and 363 genes showing up- and down-regulation, respectively, were obtained. Upon GO functional enrichment, 183, 252, and 21 specific GO terms in biological process, molecular function and cellular component were identified, respectively. Eight KEGG pathways including "Microbial metabolism in diverse environments", " S. aureus infection", and "Monobactam biosynthesis" were significantly enriched. In addition, "beta-lactam resistance" pathway was also highly enriched. In ampicillin-induced biofilm, the significant up-regulation of genes encoding multidrug resistance efflux pump AbcA, penicillin binding proteins PBP1, PBP1a/2, and PBP3, and antimicrobial resistance proteins VraF, VraG, Dlt, and Aur indicated the positive response of S. aureus to ampicillin. The up-regulation of genes encoding surface proteins ClfB, IsdA, and SasG and genes ( cap5B and cap5C ) which promote the adhesion of S. aureus in ampicillin induced biofilm might explain the enhanced biofilm viability and biomass.

Lactobacillus brevis is a major hop-resistance bacterium which poses significant challenge for the brewing industry, mainly due to the difficulty or incapability in detection by routine culturing methodology and its beer spoilage ability.This study aimed at investigating the VBNC state of a hop-resistance strain, L. brevis BM-LB13908. The culturable, total and viable numbers of L. brevis cells were calculated by MRS agar plate counting, acridine orange direct count (AODC) method and Live/Dead BacLight bacterial viability kit with fluorescence microscope. VBNC formation was induced by 189 ± 5.7 days under low-temperature storage or 27 ± 1.2 subcultures by continuous passage in beer, and VBNC cells induced by both strategies were recovered by adding catalase. In addition, insignificant difference in beer-spoilage ability was found in 3 states of L. brevis , including logarithmic growing, VBNC and recovered cells. This is the first study on the formation of VBNC state for L. brevis and beer-spoilage ability of both VBNC and recovered cells, which indicate L. brevis strain could cause beer spoilage without being detected by routine methodologies. The results derived from this study may support further study on L. brevis and other hop-resistance bacteria, and guidance on beer spoilage prevention and control, such as improvement for brewers on the microbiological quality control by using the improved culture method with catalase supplementation.

Occasional beer spoilage incidents caused by false-negative isolation of lactic acid bacteria (LAB) in the viable but non-culturable (VBNC) state, result in significant profit loss and pose a major concern in the brewing industry. In this study, both culturable and VBNC cells of an individual Lactobacillus harbinensis strain BM-LH14723 were identified in one spoiled beer sample by genome sequencing, with the induction and resuscitation of VBNC state for this strain further investigated. Formation of the VBNC state was triggered by low-temperature storage in beer (175 ± 1.4 days) and beer subculturing (25 ± 0.8 subcultures), respectively, and identified by both traditional staining method and PMA-PCR. Resuscitated cells from the VBNC state were obtained by addition of catalase rather than temperature upshift, changing medium concentration, and adding other chemicals, and both VBNC and resuscitated cells retained similar beer-spoilage capability as exponentially growing cells. In addition to the first identification of both culturable and VBNC cells of an individual L. harbinensis strain from spoiled beer, this study also for the first time reported the VBNC induction and resuscitation, as well as verification of beer-spoilage capability of VBNC and resuscitated cells for the L. harbinensis strain. Genes in association with VBNC state were also identified by the first genome sequencing of beer spoilage L. harbinensis. The results derived from this study suggested the contamination and spoilage of beer products by VBNC and resuscitated L. harbinensis strain BM-LH14723.

Inactivation of sterol Δ 5,6 -desaturase (Erg3p) in the prevalent fungal pathogen Candida albicans is one of several mechanisms that can confer resistance to the azole antifungal drugs. However, loss of Erg3p activity is also associated with deficiencies in stress tolerance, invasive hyphal growth, and attenuated virulence in a mouse model of disseminated infection. This may explain why relatively few erg3 -deficient strains have been reported among azole-resistant clinical isolates. In this study, we examined the consequences of Erg3p inactivation upon C. albicans pathogenicity and azole susceptibility in mouse models of mucosal and disseminated infection. While a C. albicans erg3 Δ/Δ mutant was unable to cause lethality in the disseminated model, it induced pathology in a mouse model of vaginal infection. The erg3 Δ/Δ mutant was also more resistant to fluconazole treatment than the wild type in both models of infection. Thus, complete loss of Erg3p activity confers azole resistance but also niche-specific virulence deficiencies. Serendipitously, we discovered that loss of azole-inducible ERG3 transcription (rather than complete inactivation) is sufficient to confer in vitro fluconazole resistance, without compromising C. albicans stress tolerance, hyphal growth, or pathogenicity in either mouse model. It is also sufficient to confer fluconazole resistance in the mouse vaginal model, but not in the disseminated model of infection, and thus confers niche-specific azole resistance without compromising C. albicans pathogenicity at either site. Collectively, these results establish that modulating Erg3p expression or activity can have niche-specific consequences on both C. albicans pathogenicity and azole resistance. IMPORTANCE While conferring resistance to the azole antifungals in vitro , loss of sterol Δ 5,6 -desaturase (Erg3p) activity has also been shown to reduce C. albicans pathogenicity. Accordingly, it has been presumed that this mechanism may not be significant in the clinical setting. The results presented here challenge this assumption, revealing a more complex relationship between Erg3p activity, azole resistance, C. albicans pathogenicity, and the specific site of infection. Most importantly, we have shown that even modest changes in ERG3 transcription are sufficient to confer azole resistance without compromising C. albicans fitness or pathogenicity. Given that previous efforts to assess the importance of ERG3 as a determinant of clinical azole resistance have focused almost exclusively on detecting null mutants, its role may have been grossly underestimated. On the basis of our results, a more thorough investigation of the contribution of the ERG3 gene to azole resistance in the clinical setting is warranted., (Copyright © 2018 Luna-Tapia et al.)

Background: Carbapenem-resistant Gram-negative bacilli (GNB) have become an important cause of nosocomial infections of hospitalized patients., Methods: To investigate the microbial infection patterns and molecular epidemiology characteristics of the carbapenem-resistant GNB isolates from a long-term hospitalized patient, antimicrobial susceptibility testing, phenotypic screening test for carbapenemase production, PCR screening and DNA sequencing of carbapenemase genes, repetitive extragenic palindromic sequence-based PCR (REP-PCR), multilocus sequencing typing (MLST) and genetic environment analysis were performed., Results: Twelve strains with carbapenemase genes were detected from 63 carbapenem-resistant isolates, including two bla IMP-25 -carrying Pseudomonas aeruginosa, one bla NDM-1 -carrying Citrobacter freundii, three bla NDM-1 -carrying Klebsiella pneumoniae and six bla KPC-2 -carrying K. pneumoniae. Only the bla NDM-1 genes were successfully transferred from three K. pneumoniae strains to Escherichia coli C600 by conjugation. Genetic environment of bla IMP-25 , bla NDM-1 and bla KPC-2 genes in our study were consistent with previous reports. Molecular typing of K. pneumoniae performed by MLST revealed that most of the isolates belonged to ST11. bla NDM-1 -carrying K. pneumoniae sequencing type 1416 was first reported in our study., Conclusions: Carbapenem-resistant GNB are common pathogens during long-term hospitalization, and ST11 bla KPC-2 -carrying K. pneumoniae is the dominant bacterium in our study. Colonization and horizontal transmission of resistance by plasmids of carbapenem-resistant GNB have increased the risks of persistent infection and mortality of long-term hospitalized patients., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objectives: We previously described the novel qnrVC6 and bla IMP-45 carrying megaplasmid pBM413. This study aimed to investigate the complete genome of multidrug-resistance P. aeruginosa Guangzhou-Pae617, a clinical isolate from the sputum of a patient who was suffering from respiratory disease in Guangzhou, China., Methods: The genome was sequenced using Illumina Hiseq 2500 and PacBio RS II sequencers and assembled de novo using HGAP. The genome was automatically and manually annotated., Results: The genome of P. aeruginosa Guangzhou-Pae617 is 6,430,493 bp containing 5881 predicted genes with an average G + C content of 66.43%. The genome showed high similarity to two new sequenced P. aeruginosa strains isolated from New York, USA. From the whole genome sequence, we identified a type IV pilin, two large prophages, 15 antibiotic resistant genes, 5 genes involved in the "Infectious diseases" pathways, and 335 virulence factors., Conclusions: The antibiotic resistance and virulence factors in the genome of P. aeruginosa strain Guangzhou-Pae617 were identified by complete genomic analysis. It contributes to further study on antibiotic resistance mechanism and clinical control of P. aeruginosa., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A plasmid pCY-CTX carrying a phage-like backbone from an extensively drug-resistant Enterobacter cloacae strain Guangzhou-ECL001 (previously known as CY01) was identified in this study. By Illumina MiSeq 2 × 250-bp paired-end sequencing, de novo assembly, and PCR, full sequence of pCY-CTX was obtained. Plasmid pCY-CTX was a circular plasmid with a length of 116,700 bp, harboring 136 putative open reading frames with the average G + C content of 50.8%. The backbone of pCY-CTX showed high identity to previously reported phage-like plasmid pHCM2 and phage SSU5. In addition, pCY-CTX contained a distinctive ISEcp1-mediated Tn2 region with two resistance genes bla TEM-1 and bla CTX-M-3 . Transposition unit "ISEcp1- bla CTX-M-3 - orf477" was inserted into the Tn2 structure, dividing Tn2 into two parts. This represents the first identification of a plasmid carrying a phage-like backbone and a distinctive ISEcp1-mediated Tn2 region within bla TEM-1 and bla CTX-M-3 in clinical E. cloacae. The finding of phage-like regions located in plasmids provides a new perspective in gene transfer associated with antimicrobial resistance.

For over 3 decades, investigators have studied the pathogenesis of vulvovaginal candidiasis (VVC) and recurrent VVC (RVVC) through clinical studies and animal models. While there was considerable consensus that susceptibility was not associated with any apparent deficiencies in adaptive immunity, protective immune mechanisms and the role of innate immunity remained elusive. It was not until an innovative live-challenge design was conducted in women that a fuller understanding of the natural history of infection/disease was achieved. These studies revealed that symptomatic infection is associated with recruitment of polymorphonuclear neutrophils (PMNs) into the vaginal lumen. Subsequent studies in the established mouse model demonstrated that infiltrating PMNs were incapable of reducing the fungal burden, which supported the hypothesis that VVC/RVVC was an immunopathology, whereby Candida and the host response drive symptomatic disease. Further studies in mice revealed the requirement for C. albicans hyphae and identified pattern recognition receptors (PRRs) and proinflammatory mediators responsible for the PMN response, all of which are critical pieces of the immunopathogenesis. However, a mechanism explaining PMN dysfunction at the vaginal mucosa remained an enigma. Ultimately, by employing mouse strains resistant or susceptible to chronic VVC, it was determined that heparan sulfate (HS) in the vaginal environment of susceptible mice serves as a competitive ligand for Mac-1 on PMNs, which effectively renders the PMNs incapable of binding to Candida to initiate killing. Hence, the outcome of symptomatic VVC/RVVC is postulated to be dependent on a PMN-mediated immunopathogenic response involving HS that effectively places the neutrophils in a state of functional anergy., (Copyright © 2018 American Society for Microbiology.)

Unlike other forms of candidiasis, vulvovaginal candidiasis, caused primarily by the fungal pathogen Candida albicans , is a disease of immunocompetent and otherwise healthy women. Despite its prevalence, the fungal factors responsible for initiating symptomatic infection remain poorly understood. One of the hallmarks of vaginal candidiasis is the robust recruitment of neutrophils to the site of infection, which seemingly do not clear the fungus, but rather exacerbate disease symptomatology. Candidalysin, a newly discovered peptide toxin secreted by C. albicans hyphae during invasion, drives epithelial damage, immune activation, and phagocyte attraction. Therefore, we hypothesized that Candidalysin is crucial for vulvovaginal candidiasis immunopathology. Anti- Candida immune responses are anatomical-site specific, as effective gastrointestinal, oral, and vaginal immunities are uniquely compartmentalized. Thus, we aimed to identify the immunopathologic role of Candidalysin and downstream signaling events at the vaginal mucosa. Microarray analysis of C. albicans -infected human vaginal epithelium in vitro revealed signaling pathways involved in epithelial damage responses, barrier repair, and leukocyte activation. Moreover, treatment of A431 vaginal epithelial cells with Candidalysin induced dose-dependent proinflammatory cytokine responses (including interleukin 1α [IL-1α], IL-1β, and IL-8), damage, and activation of c-Fos and mitogen-activated protein kinase (MAPK) signaling, consistent with fungal challenge. Mice intravaginally challenged with C. albicans strains deficient in Candidalysin exhibited no differences in colonization compared to isogenic controls. However, significant decreases in neutrophil recruitment, damage, and proinflammatory cytokine expression were observed with these strains. Our findings demonstrate that Candidalysin is a key hypha-associated virulence determinant responsible for the immunopathogenesis of C. albicans vaginitis., (Copyright © 2018 American Society for Microbiology.)

To evaluate the cariogenic properties of almond milk beverages, 6 almond milks, along with soy and whole bovine milk, were analyzed for their abilities to support Streptococcus mutans biofilm formation and acid production, and their capacity to buffer changes in pH. Biofilm formation by S. mutans was analyzed using an in vitro 96-well plate model and measured by crystal violet staining. Acid production by S. mutans was evaluated by a colorimetric L-lactate assay and pH measurement of bacterial cultures. Buffering capacity was assessed by a pH titration assay. Soy milk supported the most biofilm growth, while the least was observed with unsweetened almond milk (both p < 0.001). Among almond milks, sucrose-sweetened milk led to the highest level of biofilm formation (p < 0.001), while the least was observed with unsweetened milk (p < 0.05). Sucrose-sweetened almond milk yielded the lowest pH (4.56 ± 0.66), followed by soy milk and bovine milk; the highest pH was with unsweetened almond milk (6.48 ± 0.5). When analyzed by pH titration, the unsweetened almond milk displayed the weakest buffering capacity while bovine milk showed the highest (p < 0.001). These results suggest that the almond milk beverages, except those that are sweetened with sucrose, possess limited cariogenic properties, while soy milk exhibits the most cariogenic potential. As milk alternatives become increasingly popular, dentists must counsel their patients that almond milks, especially sucrose-sweetened varieties, have cariogenic potential. For patients who are lactose-intolerant or suffer from milk allergy, almond milks may be a better alternative than soy-based products., (© 2017 S. Karger AG, Basel.)

This study aimed to characterise a novel multidrug resistance megaplasmid carrying qnrVC6 and bla IMP-45 from Pseudomonas aeruginosa strain Guangzhou-Pae617 isolated from a patient hospitalised in Guangzhou, China, in 2012. The plasmid pBM413 has a length of 423 017 bp and an average G + C content of 56.41%. A qnrVC6 gene flanked by two copies of insertion sequence (IS) elements ISCR1, a multiresistance class 1 integron In786 containing aacA4-bla IMP-45 -bla OXA-1 -catB3 cassettes, an armA gene, and an aphA7 gene flanked by two copies of IS26 were identified. To our knowledge, this is the first identification of a qnrVC6 gene in P. aeruginosa., (Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

This study aimed to investigate the viable but nonculturable (VBNC) state and genomic features of a beer-spoilage strain, Lactobacillus caseiBM-LC14617. Induction on the VBNC state of L. casei strain BM-LC14617 was conducted by both low-temperature storage and continuous passage in beer, and formation of VBNC state was detected after 196 ± 3.3 days and 32 ± 1.6 subcultures, respectively. Resuscitation of VBNC cells was successfully induced by addition of catalase, and culturable, VBNC, and resuscitated cells shared similar beer-spoilage capability. Whole genome sequencing was performed, and out of a total of 3,964 predicted genes, several potential VBNC and beer-spoilage-associated genes were identified. L. casei is capable of entering into and resuscitating from the VBNC state and possesses beer-spoilage capability. The genomic characterization yield insightful elucidation of VBNC state for L. casei. This study represents the first evidence on VBNC state induction of L. casei and beer-spoilage capability of VBNC and resuscitated cells. Also, this is the first genomic characterization of L. casei as a beer-spoilage bacterium. The current study may aid in further study on L. casei and other beer-spoilage bacteria, and guide the prevention and control of beer spoilage., (© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

The secreted aspartyl proteinases of Candida albicans have long been implicated in virulence at the mucosal surface, including contributions to colonization and immunopathogenesis during vulvovaginal candidiasis. In an effort to disentangle hypha-associated virulence factor regulation from morphological transition, the purpose of this study was to determine if overexpression of SAP2 or SAP5 in an efg1 Δ/Δ cph1 Δ/Δ mutant could restore the capacity to cause immunopathology during murine vaginitis to this avirulent hypofilamentous strain. Two similar yet distinct genetic approaches were used to construct expression vectors to achieve SAP overexpression, and both genetic and functional assays confirmed elevated SAP activity in transformed strains. Similar to previous findings, intravaginal challenge of C57BL/6 mice with hypha-defective strains attained high levels of mucosal colonization but failed to induce robust vaginal immunopathology (neutrophil recruitment, interleukin-1β [IL-1β] secretion, and lactate dehydrogenase release) compared to that with the hypha-competent control. Moreover, constitutive expression of SAP2 or SAP5 in two distinct sets of such strains did not elicit immunopathological markers at levels above those observed during challenge with isogenic empty vector controls. Therefore, these results suggest that the physiological contributions of SAPs to vaginal immunopathology require hypha formation, other hypha-associated factors, or genetic interaction with EFG1 and/or CPH1 to cause symptomatic infection. Additionally, the outlined expression strategy and strain sets will be useful for decoupling other downstream morphogenetic factors from hyphal growth., (Copyright © 2017 American Society for Microbiology.)