Enhancing probiotic impact: engineering Saccharomyces boulardii for optimal acetic acid production and gastric passage tolerance.

Saccharomyces boulardii has been a subject of growing interest due to its potential as a probiotic microorganism with applications in gastrointestinal health, but the molecular cause for its probiotic potency has remained elusive. The recent discovery that S. boulardii contains unique mutations causing high acetic acid accumulation and inhibition of bacterial growth provides a possible clue. The natural S. boulardii isolates Sb.P and Sb.A are homozygous for the recessive mutation whi2S270* and accumulate unusually high amounts of acetic acid, which strongly inhibit bacterial growth. However, the homozygous whi2S270* mutation also leads to acetic acid sensitivity and acid sensitivity in general. In the present study, we have constructed a new S. boulardii strain, derived from the widely therapeutically used CMCN I-745 strain (isolated from the pharmaceutical product Enterol), producing even higher levels of acetic acid while keeping the same tolerance toward low pH as the parent Enterol (ENT) strain. This newly engineered strain, named ENT3, has a homozygous deletion of ACH1 and strong overexpression of ALD4. It is also able to accumulate much higher acetic acid concentrations when growing on low glucose levels, in contrast to the ENT wild-type and Sb.P strains. Moreover, we show the antimicrobial capacity of ENT3 against gut pathogens in vitro and observed that higher acetic acid production might correlate with better persistence in the gut in healthy mice. These findings underscore the possible role of the unique acetic acid production and its potential for improvement of the probiotic action of S. boulardii.IMPORTANCESuperior variants of the probiotic yeast Saccharomyces boulardii produce high levels of acetic acid, which inhibit the growth of bacterial pathogens. However, these strains also show increased acid sensitivity, which can compromise the viability of the cells during their passage through the stomach. In this work, we have developed by genetic engineering a variant of Saccharomyces boulardii that produces even higher levels of acetic acid and does not show enhanced acid sensitivity. We also show that the S. boulardii yeasts with higher acetic acid production persist longer in the gut, in agreement with a previous work indicating competition between probiotic yeast and bacteria for residence in the gut.

New biomarkers underlying acetic acid tolerance in the probiotic yeast Saccharomyces cerevisiae var. boulardii.

Evolutionary engineering experiments, in combination with omics technologies, revealed genetic markers underpinning the molecular mechanisms behind acetic acid stress tolerance in the probiotic yeast Saccharomyces cerevisiae var. boulardii. Here, compared to the ancestral Ent strain, evolved yeast strains could quickly adapt to high acetic acid levels (7 g/L) and displayed a shorter lag phase of growth. Bioinformatic-aided whole-genome sequencing identified genetic changes associated with enhanced strain robustness to acetic acid: a duplicated sequence in the essential endocytotic PAN1 gene, mutations in a cell wall mannoprotein (dan4Thr192del), a lipid and fatty acid transcription factor (oaf1Ser57Pro) and a thiamine biosynthetic enzyme (thi13Thr332Ala). Induction of PAN1 and its associated endocytic complex SLA1 and END3 genes was observed following acetic acid treatment in the evolved-resistant strain when compared to the ancestral strain. Genome-wide transcriptomic analysis of the evolved Ent acid-resistant strain (Ent ev16) also revealed a dramatic rewiring of gene expression among genes associated with cellular transport, metabolism, oxidative stress response, biosynthesis/organization of the cell wall, and cell membrane. Some evolved strains also displayed better growth at high acetic acid concentrations and exhibited adaptive metabolic profiles with altered levels of secreted ethanol (4.0-6.4% decrease), glycerol (31.4-78.5% increase), and acetic acid (53.0-60.3% increase) when compared to the ancestral strain. Overall, duplication/mutations and transcriptional alterations are key mechanisms driving improved acetic acid tolerance in probiotic strains. We successfully used adaptive evolutionary engineering to rapidly and effectively elucidate the molecular mechanisms behind important industrial traits to obtain robust probiotic yeast strains for myriad biotechnological applications. KEY POINTS: •Acetic acid adaptation of evolutionary engineered robust probiotic yeast S. boulardii •Enterol ev16 with altered genetic and transcriptomic profiles survives in up to 7 g/L acetic acid •Improved acetic acid tolerance of S. boulardii ev16 with mutated PAN1, DAN4, OAF1, and THI13 genes.

Oteseconazole: a long-awaited diversification of the antifungal arsenal to manage recurrent vulvovaginal candidiasis (RVVC).

INTRODUCTION: Recurrent vulvovaginal candidiasis (RVVC) affects women worldwide and has far-reaching implications for a patient's quality of life. For decades, maintenance treatment using the azole antifungal fluconazole was the preferred treatment. Although efficient in controlling the symptoms, the development of azole resistance and high rates of recurrence after therapy cessation have emerged as significant limitations. Nevertheless, persistent efforts have delivered novel treatment options. Oteseconazole (VT-1161), marketed as VIVJOA, is an oral, tetrazole antifungal with unprecedented specificity toward the fungal lanosterol 14α-demethylase. AREAS COVERED: We reviewed literature data on oteseconazole with a focus on the management of RVVC. EXPERT OPINION: Therapeutic options for RVVC are limited, and novel, innovative approaches are needed to treat this debilitating condition. These therapies need to be well-tolerated and prevent RVVC recurrence. The available clinical data show excellent safety and efficacy, with an unprecedentedly low recurrence rate. However, we believe health-care providers should be mindful to monitor for the development of resistance, as this may result in treatment failure. Further, the availability and cost may, like for most novel drugs, affect the widespread clinical implementation of VIVJOA. Altogether, we are convinced that VIVJOA is a significant advance in RVVC management.

Sources of Antifungal Drugs.

Due to their eukaryotic heritage, the differences between a fungal pathogen's molecular makeup and its human host are small. Therefore, the discovery and subsequent development of novel antifungal drugs are extremely challenging. Nevertheless, since the 1940s, researchers have successfully uncovered potent candidates from natural or synthetic sources. Analogs and novel formulations of these drugs enhanced the pharmacological parameters and improved overall drug efficiency. These compounds ultimately became the founding members of novel drug classes and were successfully applied in clinical settings, offering valuable and efficient treatment of mycosis for decades. Currently, only five different antifungal drug classes exist, all characterized by a unique mode of action; these are polyenes, pyrimidine analogs, azoles, allylamines, and echinocandins. The latter, being the latest addition to the antifungal armamentarium, was introduced over two decades ago. As a result of this limited arsenal, antifungal resistance development has exponentially increased and, with it, a growing healthcare crisis. In this review, we discuss the original sources of antifungal compounds, either natural or synthetic. Additionally, we summarize the existing drug classes, potential novel candidates in the clinical pipeline, and emerging non-traditional treatment options.

In Vitro Assessment of Azole and Amphotericin B Susceptibilities of Malassezia spp. Isolated from Healthy and Lesioned Skin.

Malassezia yeasts have recently gained medical importance as emerging pathogens associated with a wide range of dermatological and systemic infections. Since standardized methods for in vitro antifungal susceptibility testing have not yet been established for Malassezia spp., related diseases are always treated empirically. As a result, a high rate of recurrence and decreased antifungal susceptibility have appeared. Thus, the aims of the study were to assess and analyze the in vitro susceptibility of Malassezia isolated from pityriasis versicolor (PV) lesions and healthy controls. A total of 58 Malassezia strains isolated from PV patients and healthy controls were tested. In vitro antifungal susceptibility testing was conducted using the CLSI broth microdilution with some modifications. Candida spp. criteria established in accordance with CLSI guidelines were used for data interpretation. Ketoconazole and posaconazole seemed to be the most effective molecules against Malassezia species. However, considerable percentages of itraconazole, fluconazole, and amphotericin B ''resistant'' strains (27.6%, 29.3%, and 43.1%, respectively) were revealed in this study. Malassezia furfur, M. sympodialis, and M. globosa showed different susceptibility profiles to the drugs tested. These results emphasize the importance of accurately identifying and evaluating the antifungal susceptibility of Malassezia species in order to guide a specific and effective treatment regimen.

The Role of Fatty Acid Metabolites in Vaginal Health and Disease: Application to Candidiasis.

Although the vast majority of women encounters at least one vaginal infection during their life, the amount of microbiome-related research performed in this area lags behind compared to alternative niches such as the intestinal tract. As a result, effective means of diagnosis and treatment, especially of recurrent infections, are limited. The role of the metabolome in vaginal health is largely elusive. It has been shown that lactate produced by the numerous lactobacilli present promotes health by limiting the chance of infection. Short chain fatty acids (SCFA) have been mainly linked to dysbiosis, although the causality of this relationship is still under debate. In this review, we aim to bring together information on the role of the vaginal metabolome and microbiome in infections caused by Candida. Vulvovaginal candidiasis affects near to 70% of all women at least once in their life with a significant proportion of women suffering from the recurrent variant. We assess the role of fatty acid metabolites, mainly SCFA and lactate, in onset of infection and virulence of the fungal pathogen. In addition, we pinpoint where lack of research limits our understanding of the molecular processes involved and restricts the possibility of developing novel treatment strategies.

Molecular Elucidation of Riboflavin Production and Regulation in Candida albicans, toward a Novel Antifungal Drug Target.

Candida albicans is a major cause of fungal infections, both superficial and invasive. The economic costs as well as consequences for patient welfare are substantial. Only a few treatment options are available due to the high resemblance between fungal targets and host molecules, as both are eukaryotes. Riboflavin is a yellow pigment, also termed vitamin B2 Unlike animals, fungi can synthesize this essential component themselves, thereby leading us to appreciate that targeting riboflavin production is a promising novel strategy against fungal infections. Here, we report that the GTP cyclohydrolase encoded by C. albicansRIB1 (CaRIB1) is essential and rate-limiting for production of riboflavin in the fungal pathogen. We confirm the high potential of CaRib1 as an antifungal drug target, as its deletion completely impairs in vivo infectibility by C. albicans in model systems. Furthermore, the stimulating effect of iron deprivation and PKA activation on riboflavin production seems to involve CaRib1 and the upstream transcription factor CaSef1. Gathering insights in the synthesis mechanism of riboflavin in pathogenic fungi, like C. albicans, will allow us to design a novel strategy and specifically target this process to combat fungal infections.IMPORTANCECandida albicans is an important fungal pathogen causing common superficial infections as well as invasive diseases with an extremely high morbidity and mortality. Antifungal therapies are limited in efficiency and availability. In this research, we describe the regulation of riboflavin production in C. albicans Since riboflavin biosynthesis is essential to this organism, we can appreciate that targeting it would be a promising new strategy to combat these fungal infections. We provide evidence that one particular enzyme in the production process, CaRib1, would be most promising as an antifungal drug target, as it plays a central role in regulation and proves to be essential in a mouse model of systemic infection.

Unique genetic basis of the distinct antibiotic potency of high acetic acid production in the probiotic yeast Saccharomyces cerevisiae var. boulardii.

The yeast Saccharomyces boulardii has been used worldwide as a popular, commercial probiotic, but the basis of its probiotic action remains obscure. It is considered conspecific with budding yeast Saccharomyces cerevisiae, which is generally used in classical food applications. They have an almost identical genome sequence, making the genetic basis of probiotic potency in S. boulardii puzzling. We now show that S. boulardii produces at 37°C unusually high levels of acetic acid, which is strongly inhibitory to bacterial growth in agar-well diffusion assays and could be vital for its unique application as a probiotic among yeasts. Using pooled-segregant whole-genome sequence analysis with S. boulardii and S. cerevisiae parent strains, we succeeded in mapping the underlying QTLs and identified mutant alleles of SDH1 and WHI2 as the causative alleles. Both genes contain a SNP unique to S. boulardii (sdh1 F317Y and whi2 S287*) and are fully responsible for its high acetic acid production. S. boulardii strains show different levels of acetic acid production, depending on the copy number of the whi2 S287* allele. Our results offer the first molecular explanation as to why S. boulardii could exert probiotic action as opposed to S. cerevisiae They reveal for the first time the molecular-genetic basis of a probiotic action-related trait in S. boulardii and show that antibacterial potency of a probiotic microorganism can be due to strain-specific mutations within the same species. We suggest that acquisition of antibacterial activity through medium acidification offered a selective advantage to S. boulardii in its ecological niche and for its application as a probiotic.

Polygenic analysis and targeted improvement of the complex trait of high acetic acid tolerance in the yeast Saccharomyces cerevisiae.

BACKGROUND: Acetic acid is one of the major inhibitors in lignocellulose hydrolysates used for the production of second-generation bioethanol. Although several genes have been identified in laboratory yeast strains that are required for tolerance to acetic acid, the genetic basis of the high acetic acid tolerance naturally present in some Saccharomyces cerevisiae strains is unknown. Identification of its polygenic basis may allow improvement of acetic acid tolerance in yeast strains used for second-generation bioethanol production by precise genome editing, minimizing the risk of negatively affecting other industrially important properties of the yeast. RESULTS: Haploid segregants of a strain with unusually high acetic acid tolerance and a reference industrial strain were used as superior and inferior parent strain, respectively. After crossing of the parent strains, QTL mapping using the SNP variant frequency determined by pooled-segregant whole-genome sequence analysis revealed two major QTLs. All F1 segregants were then submitted to multiple rounds of random inbreeding and the superior F7 segregants were submitted to the same analysis, further refined by sequencing of individual segregants and bioinformatics analysis taking into account the relative acetic acid tolerance of the segregants. This resulted in disappearance in the QTL mapping with the F7 segregants of a major F1 QTL, in which we identified HAA1, a known regulator of high acetic acid tolerance, as a true causative allele. Novel genes determining high acetic acid tolerance, GLO1, DOT5, CUP2, and a previously identified component, VMA7, were identified as causative alleles in the second major F1 QTL and in three newly appearing F7 QTLs, respectively. The superior HAA1 allele contained a unique single point mutation that significantly improved acetic acid tolerance under industrially relevant conditions when inserted into an industrial yeast strain for second-generation bioethanol production. CONCLUSIONS: This work reveals the polygenic basis of high acetic acid tolerance in S. cerevisiae in unprecedented detail. It also shows for the first time that a single strain can harbor different sets of causative genes able to establish the same polygenic trait. The superior alleles identified can be used successfully for improvement of acetic acid tolerance in industrial yeast strains.