Effect of Acetic Acid and Lactic Acid at Low pH in Growth and Azole Resistance of Candida albicans and Candida glabrata

Successful colonization of the acidic vaginal niche by C. glabrata and C. albicans depends on their ability to cope with the presence of lactic and acetic acids produced by commensal microbiota. As such, the inhibitory effect of these acids at a low pH in growth of C. glabrata and C. albicans was investigated. The effect of the presence of these organic acids in tolerance of the two Candida species to azoles used in treatment of vaginal infections was also investigated including eventual synergistic effects. Under the different experimental conditions tested lactic acid exerted no significant inhibitory effect against C. albicans or C. glabrata, contrasting with the generalized impression that the production of this acid is on the basis of the protective effect exerted by vaginal lactobacilii. Differently, C. glabrata and C. albicans exhibited susceptibility to acetic acid, more prominent at lower pHs and stronger for the latter species. Synergism between acetic acid and azoles was observed both for C. albicans and C. glabrata, while lactic acid-azole synergism was only efficient against C. albicans. Altogether our in vitro results indicate that tolerance to acetic acid at a low pH may play a more relevant role than tolerance to lactic acid in determining competitiveness in the vaginal tract of C. albicans and C. glabrata including under azole stress. Treatment of vaginal candidiasis with azoles may depend on the level of acetic and lactic acids present and improvements could be achieved synergizing the azole with these acids

Genome-wide screening of Saccharomyces cerevisiae genes required to foster tolerance towards industrial wheat straw hydrolysates

The presence of toxic compounds derived from biomass pre-treatment in fermentation media represents an important drawback in second-generation bio-ethanol production technology and overcoming this inhibitory effect is one of the fundamental challenges to its industrial production. The aim of this study was to systematically identify, in industrial medium and at a genomic scale, the Saccharomyces cerevisiae genes required for simultaneous and maximal tolerance to key inhibitors of lignocellulosic fermentations. Based on the screening of EUROSCARF haploid mutant collection, 242 and 216 determinants of tolerance to inhibitory compounds present in industrial wheat straw hydrolysate (WSH) and in inhibitor-supplemented synthetic hydrolysate were identified, respectively. Genes associated to vitamin metabolism, mitochondrial and peroxisomal functions, ribosome biogenesis and microtubule biogenesis and dynamics are among the newly found determinants of WSH resistance. Moreover, PRS3, VMA8, ERG2, RAV1 and RPB4 were confirmed as key genes on yeast tolerance and fermentation of industrial WSH.The authors thank Juan Carlos Parajo and Hector Ruiz for assistance in the pre-treatment of lignocellulose biomass. Research described in this article was financially supported by FEDER and "Fundacao para a Ciencia e a Tecnologia" (FCT) (Contracts PEst-OE/EQB/LA0023/2011, PTDC/BIO/66151/2006, PTDC/AGR-ALI/102608/2008 and ERA-IB/0002/2010 and PhD grant (SFRH/BD/64776/2009) to FP)

Genome-wide screening of Saccharomyces cerevisiae genes required to foster tolerance towards inhibitory compounds in industrial biomass hydrolysates

The understanding of the determinants of yeast tolerance to inhibitory compounds present in fermentation media at the genetic level is of essential importance for the improvement of second generation bio-ethanol conversion technology. The aim of this study was to systematically identify, at a genomic scale, the Saccharomyces cerevisiae genes required for simultaneous and maximal tolerance to key inhibitors derived from lignocellulose biomass pre-treatment. Based on the screening of the EUROSCARF haploid mutant collection, 242 and 216 determinants of yeast resistance to inhibitory compounds present in industrial wheat straw hydrolysate (WSH) and in inhibitor-supplemented synthetic hydrolysate (SH) were identified, respectively. Twenty-two mutants with deleted genes involved in Oxidative stress response, Lipid Metabolism, Aminoacid metabolism, Vacuolar acidification, Intracellular trafficking and protein sorting, Transcription machinery and RNA processing and Mitochondrial function showed a strong susceptibility phenotype in both WSH and SH, 8 of them being for the first time identified as conferring resistance to lignocellulose-derived inhibitors. The intersection of our WSH and SH datasets and those obtained in previous genome-wide studies on single chemical stress resistance, together with results obtained during comparative fermentative performance analysis [1], provided data for further evaluation of the key genes involved in global adaptation to toxic biomass hydrolysates. This study expands our understanding of the genes and underlying molecular mechanisms that are directly involved in yeast response to the multiple stresses occurring during lignocellulose fermentations under industrially relevant conditions

Effect of progesterone on Candida albicans biofilm formation under acidic conditions: a transcriptomic analysis

Supplementary material related to this article can be found, in the online version, at doi: https://doi.org/10.1016/j.ijmm.2020.151414.Vulvovaginal candidiasis (VVC) caused by Candida albicans is a common disease worldwide. A very important C. albicans virulence factor is its ability to form biofilms on epithelium and/or on intrauterine devices promoting VVC. It has been shown that VVC has a hormonal dependency and that progesterone affects virulence traits of C. albicans cells. To understand how the acidic environment (pH 4) and progesterone (either alone and in combination) modulate C. albicans response during formation of biofilm, a transcriptomic analysis was performed together with characterization of the biofilm properties. Compared to planktonic cells, acidic biofilm-cells exhibited major changes in their transcriptome, including modifications in the expression of 286 genes that were not previously associated with biofilm formation in C. albicans. The vast majority of the genes up-regulated in the acidic biofilm cells (including those uniquely identified in our study) are known targets of Sfl1, and consistently, Sfl1 deletion is herein shown to impair the formation of acidic biofilms (pH4). Under the acidic conditions used, the presence of progesterone reduced C. albicans biofilm biomass and structural cohesion. Transcriptomic analysis of biofilms developed in the presence of progesterone led to the identification of 65 down-regulated genes including, among others, the regulator Tec1 and several of its target genes, suggesting that the function of this transcription factor is inhibited by the presence of the hormone. Additionally, progesterone reduced the susceptibility of biofilm cells to fluconazole, consistent with an up-regulation of efflux pumps. Overall, the results of this study show that progesterone modulates C. albicans biofilm formation and genomic expression under acidic conditions, which may have implications for C. albicans pathogenicity in the vaginal environment.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145- FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. Funding received by iBB–Institute for Bioengineering and Biosciences from FCT (UID/BIO/04565/2013), from Programa Operacional Regional de Lisboa 2020 Project No. 007317 is also acknowledged. Support from FCT to NAP through the doctoral program Applied and Environmental Microbiology (PD/BD/143026/2018) is also acknowledged. B. Gonçalves is recipient of a PhD grant supported by FCT (SFRH/BD/111645/2015). G. Butler was supported by Science Foundation Ireland (12/IA/1343).info:eu-repo/semantics/publishedVersio

Characterizing the potential of the non-conventional yeast Saccharomycodes ludwigii UTAD17 in winemaking

Non-Saccharomyces yeasts have received increased attention by researchers and winemakers, due to their particular contributions to the characteristics of wine. In this group, Saccharomycodes ludwigii is one of the less studied species. In the present study, a native S. ludwigii strain, UTAD17 isolated from the Douro wine region was characterized for relevant oenological traits. The genome of UTAD17 was recently sequenced. Its potential use in winemaking was further evaluated by conducting grape-juice fermentations, either in single or in mixed-cultures, with Saccharomyces cerevisiae, following two inoculation strategies (simultaneous and sequential). In a pure culture, S. ludwigii UTAD17 was able to ferment all sugars in a reasonable time without impairing the wine quality, producing low levels of acetic acid and ethyl acetate. The overall effects of S. ludwigii UTAD17 in a mixed-culture fermentation were highly dependent on the inoculation strategy which dictated the dominance of each yeast strain. Wines whose fermentation was governed by S. ludwigii UTAD17 presented low levels of secondary aroma compounds and were chemically distinct from those fermented by S. cerevisiae. Based on these results, a future use of this non-Saccharomyces yeast either in monoculture fermentations or as a co-starter culture with S. cerevisiae for the production of wines with greater expression of the grape varietal character and with flavor diversity could be foreseen. View Full-Textinfo:eu-repo/semantics/publishedVersio

Assessing the ability of Lactobacillus strains to counteract enterotoxigenic Escherichia coli (ETEC) infection by using a Galleria mellonella in vivo model

Enteric colibacillosis is a common disease in weanling pigs, with postweaning diarrhea (PWD) as the main symptom in piglets. It is caused by the colonization of the small intestine by enterotoxigenic strains of Escherichia coli (ETEC). Of the control strategies, antibiotics and zinc oxide have been the most effective in reducing the economic losses caused by PWD. However, concerns about antibiotic resistance have led to restrictions on the use of critically important antimicrobials in food-producing animals, and in June 2021 zinc oxide was banned in the European Union due to the environmental risks it poses. As a result, efforts are underway to develop more environmentally friendly alternatives to combat ETEC infections, such as probiotics. In this study, we evaluated the ability of three potential probiotics (Lactobacillus gasseri, L. acidophilus and L. reuteri) to reduce the ETEC infection by using a Galleria mellonella in vivo model in 2 different perspectives: co-infection (i.e. Lactobacillus + ETEC); and prophylactic strategy (i.e. prior infection with Lactobacillus for 4 h followed by ETEC infection). Survival rate and health index scores of G. mellonella were assessed at 24, 48, and 72 h post-infection. In addition, real-time PCR was also performed to determine the transcript levels of genes encoding the G. mellonella antimicrobial peptides to infer the immune response to ETEC infection. Our results suggest that a co-infection strategy was not effective in controlling ETEC infection. On the other hand, when a prophylactic strategy was used, we observed significant differences between the treated larvae and the control. Overall, we observed that L. acidophilus was able to reduce ETEC strain SP11 infection. Differences in the expression of antimicrobial peptides were also found when comparing treated and control conditions. In conclusion, specific Lactobacillus species seem to have the potential to protect against ETEC infection.This work was financially supported by the Project PTDC/CVT-CVT/4620/2021, funded by FEDER funds through COMPETE2020–Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through FCT/MCTES. It was also supported by: LA/P/0045/2020 (ALiCE), UIDB/00511/2020 and UIDP/00511/2020 (LEPABE), funded by national funds through FCT/MCTES (PIDDAC); and under the scope of the strategic funding of UIDB/04469/2020 unit (CEB). J.C. also thanks FCT for the CEEC Individual (2022.06886.CEECIND)info:eu-repo/semantics/publishedVersio

Identification of Saccharomyces cerevisiae genes involved in the resistance to multiple stresses during Very-High-Gravity and lignocellulosic biomass industrial fermentations

Most of the current processes for bioethanol production are based on the use of Very-High-Gravity (VHG) technology and the processing of lignocellulosic biomass, limited by the high osmotic pressure and ethanol concentration in the fermentation medium, and by inhibitors resulting from biomass pre-treatments, respectively. Aiming the optimization of strains for industrial bioethanol production an integrated approach was undertaken to identify genes required for simultaneous yeast resistance to different fermentation-related stresses. The integration of previous chemogenomics data was used to identify eight genes whose expression confers simultaneous resistance to high concentrations of glucose, acetic acid and ethanol, chemical stresses relevant for VHG fermentations; and eleven genes conferring simultaneous resistance to different inhibitors present during lignocellulosic fermentations. The expression of BUD31 and HPR1 lead to the increase of both ethanol yield and fermentation rate, while PHO85, VRP1 and YGL024w expression is required for maximal ethanol production in VHG fermentations. Five genes, ERG2, PRS3, RAV1, RPB4 and VMA8 were found to contribute to the maintenance of cell viability in wheat straw hydrolysate and/or for maximal fermentation rate of this substrate [1]. Moreover, the yeast disruptome was screened for strains with increased susceptibility to inhibitory compounds present in an industrial lignocellulosic hydrolysate obtained from wheat straw. With this genome-wide analysis, 42 determinants of resistance to inhibitors were identified showing a high susceptibility phenotype compared to the parental strain. The identified genes stand as preferential targets for genetic engineering manipulation to generate more robust and efficient industrial strains

Identification of candidate genes for yeast engineering to improve bioethanol production in very high gravity and lignocellulosic biomass industrial fermentations

<p>Abstract</p> <p>Background</p> <p>The optimization of industrial bioethanol production will depend on the rational design and manipulation of industrial strains to improve their robustness against the many stress factors affecting their performance during very high gravity (VHG) or lignocellulosic fermentations. In this study, a set of <it>Saccharomyces cerevisiae </it>genes found, through genome-wide screenings, to confer resistance to the simultaneous presence of different relevant stresses were identified as required for maximal fermentation performance under industrial conditions.</p> <p>Results</p> <p>Chemogenomics data were used to identify eight genes whose expression confers simultaneous resistance to high concentrations of glucose, acetic acid and ethanol, chemical stresses relevant for VHG fermentations; and eleven genes conferring simultaneous resistance to stresses relevant during lignocellulosic fermentations. These eleven genes were identified based on two different sets: one with five genes granting simultaneous resistance to ethanol, acetic acid and furfural, and the other with six genes providing simultaneous resistance to ethanol, acetic acid and vanillin. The expression of <it>Bud31 </it>and <it>Hpr1 </it>was found to lead to the increase of both ethanol yield and fermentation rate, while <it>Pho85</it>, <it>Vrp1 </it>and <it>Ygl024w </it>expression is required for maximal ethanol production in VHG fermentations. Five genes, <it>Erg2</it>, <it>Prs3</it>, <it>Rav1</it>, <it>Rpb4 </it>and <it>Vma8</it>, were found to contribute to the maintenance of cell viability in wheat straw hydrolysate and/or the maximal fermentation rate of this substrate.</p> <p>Conclusions</p> <p>The identified genes stand as preferential targets for genetic engineering manipulation in order to generate more robust industrial strains, able to cope with the most significant fermentation stresses and, thus, to increase ethanol production rate and final ethanol titers.</p

The CgHaa1-dependent pathway mediates Candida glabrata response and tolerance to acetic acid thereby enhancing colonization of vaginal epithelium

To successfully colonize the vaginal tract Candida glabrata has to cope with various stresses including the presence of acetic acid at a low pH that is produced by the bacteria that co-colonize this niche. The genes/pathways involved in C. glabrata tolerance and response to acetic acid are largely unknown, although these are a highly interesting set of novel targets to control vaginal infections caused by this yeast. Saccharomyces cerevisae response and tolerance to acetic acid was found to be largely mediated by the ScHaa1 transcription factor [1,2,3]. In this work the involvement of CgHaa1 in C. glabrata tolerance and response to acetic acid is demonstrated. Elimination of CgHAA1 gene from C. glabrata genome dramatically increased susceptibility of this pathogenic yeast to acetic acid (30 mM at pH 4.0). Around 140 genes were found to be up-regulated, directly or indirectly, by CgHaa1 in response to acetic acid stress, based on results of a transcriptomic analysis. Functional clustering of the genes activated by CgHaa1 under acetic acid stress shows an enrichment of those involved in carbohydrate metabolism, transport, cell wall maintenance, regulation of internal pH and nucleic acid processing. At least five of the CgHaa1-regulated genes were found to increase C. glabrata tolerance to acetic acid including CgGAD1, encoding a glutamate decarboxylase; CgTPO2/3, encoding a drug efflux pump of the Major Facilitator Superfamily; CgYPS1, encoding a cell wall aspartyl protease; and CAGL0H04851 and CAGL0E03740, encoding two uncharacterized ORFs. Altogether our results are consistent with the concept that the CgHaa1- signalling pathway increases C. glabrata tolerance to acetic acid by reducing the internal accumulation of the acid and by up-regulating the activity of the plasma membrane proton pump H+-ATPase CgPma1, two essential features for a robust weak acid response. The role exerted by CgHaa1 in the ability of C. glabrata to colonize reconstituted vaginal human epithelium (RVHE) in the presence of acetic acid (30 mM at pH 4.0) was also investigated in this work. In the absence of acetic acid wild-type and DCgHaa1 mutant cells were able to colonize RVHE at a similar rate, however, in the presence of acetic acid colonization of the vaginal tissue was markedly reduced in the mutant background. The reduced colonizing capacity of DCgHaa1 mutant cells was correlated with a reduced expression of the adhesin-encoding genes EPA6, EPA7 and EPA1 and with a lower adhesiveness to the extracellular matrix proteins fibronectin and vitronectin

On the potential role of naturally occurring carboxylic organic acids as anti-infective agents:opportunities and challenges

Carboxylic organic acids are intermediates of central carbon metabolic pathways (e.g. acetic, propionic, citric, and lactic acid) long known to have potent antimicrobial potential, mainly at acidic pHs. The food industry has been leveraging those properties for years, using many of these acids as preservatives to inhibit the growth of pathogenic and/or spoilage fungal and bacterial species. A few of these molecules (the most prominent being acetic acid) have been used as antiseptics since Hippocratic medicine, mainly to treat infected wounds in patients with burns. With the growth of antibiotic therapy, the use of carboxylic acids (and other chemical antiseptics) in clinical settings lost relevance; however, with the continuous emergence of multi-antibiotic/antifungal resistant strains, the search for alternatives has intensified. This prospective article raises awareness of the potential of carboxylic acids to control infections in clinical settings, considering not only their previous exploitation in this context (which we overview) but also the positive experience of their safe use in food preservation. At a time of great concern with antimicrobial resistance and the slow arrival of new antimicrobial therapeutics to the market, further exploration of organic acids as anti-infective molecules may pave the way to more sustainable prophylactic and therapeutic approaches