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Cited by 49Antifungal resistance and susceptibilityFungal and yeast genetics researchFungal Infections and Studies

Candida glabrata susceptibility to antifungals and phagocytosis is modulated by acetate

Open access
Mota, Sandra, Alves, Rosana, Carneiro, Catarina, Silva, Sónia, Brown, Alistair J., Istel, Fabian, Kuchler, Karl, Sampaio, Paula +3 more·2015·Frontiers in Microbiology
DOI 10.3389/fmicb.2015.00919PMC PMC4560035
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Abstract

Candida glabrata is considered a major opportunistic fungal pathogen of humans. The capacity of this yeast species to cause infections is dependent on the ability to grow within the human host environment and to assimilate the carbon sources available. Previous studies have suggested that C. albicans can encounter glucose-poor microenvironments during infection and that the ability to use alternative non-fermentable carbon sources, such as carboxylic acids, contributes to the virulence of this fungus. Transcriptional studies on C. glabrata cells identified a similar response, upon nutrient deprivation. In this work, we aimed at analyzing biofilm formation, antifungal drug resistance, and phagocytosis of C. glabrata cells grown in the presence of acetic acid as an alternative carbon source. C. glabrata planktonic cells grown in media containing acetic acid were more susceptible to fluconazole and were better phagocytosed and killed by macrophages than when compared to media lacking acetic acid. Growth in acetic acid also affected the ability of C. glabrata to form biofilms. The genes ADY2a, ADY2b, FPS1, FPS2, and ATO3, encoding putative carboxylate transporters, were upregulated in C. glabrata planktonic and biofilm cells in the presence of acetic acid. Phagocytosis assays with fps1 and ady2a mutant strains suggested a potential role of FPS1 and ADY2a in the phagocytosis process. These results highlight how acidic pH niches, associated with the presence of acetic acid, can impact in the treatment of C. glabrata infections, in particular in vaginal candidiasis.

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Written from the paper’s full text.

TL;DR

In *Candida glabrata*, growth in acetic acid at pH 5.0 increased planktonic susceptibility to fluconazole and reduced biofilm biomass/viability, while also making wild-type cells more phagocytosed and killed by macrophages. Acetate exposure upregulated putative carboxylate transporter genes *ADY2a*, *ADY2b*, *FPS1*, and *ATO3*, and mutant assays suggested roles for *FPS1* and *ADY2a* in phagocytosis/killing.

Key findings

  • Planktonic *C. glabrata* ATCC2001 grown in RPMI pH 5.0 had fluconazole MIC 150 μg/ml and MFC 312.5 μg/ml; with 0.5% acetic acid pH 5.0, MIC fell to 50 μg/ml and MFC to 150 μg/ml.
  • Acetic acid reduced biofilm biomass and viable cell numbers, but fluconazole did not eradicate biofilms in either condition; significant biomass and CFU reduction occurred only at 1250 μg/ml. *ady2a* and *fps1* mutant biofilms were similar to wild type.
  • *ADY2a*, *ADY2b*, *FPS1*, and *ATO3* were upregulated in planktonic and biofilm cells with acetic acid, with *ADY2a* strongly induced and *FPS2* expressed at low levels.
  • Acetic acid-grown wild-type cells showed 14.5% of macrophages with internalized yeast versus 7.6% in RPMI, and a phagocytosis index of 14.7% versus 8.1%; killing was higher after 3 h. In acetic acid, *ady2a* cells were more resistant to macrophage killing, while *fps1* cells were more phagocytosed; TNF-α release was similar across conditions.

Methods

  • *C. glabrata* strains ATCC2001, HTL, *ady2a*, and *fps1* were grown in RPMI with or without 0.5% acetic acid at pH 5.0; MIC/MFC were determined by CLSI microdilution with fluconazole, and biofilms were assessed by crystal violet biomass, CFU counts, and SEM.
  • Gene expression was measured by qRT-PCR for *ADY2a*, *ADY2b*, *ATO3*, *FPS1*, and *FPS2*, normalized to *PGK1*; phagocytosis/killing assays used murine J774A.1 macrophages with flow cytometry, fluorescence microscopy, CFU counting, and TNF-α ELISA.
  • Statistical analysis used one-way ANOVA with Tukey multiple comparison test at 95% confidence.

Limitations

  • The paper does not state a dedicated limitations section.
  • Evident from the text: all experiments are in vitro, and macrophage work uses the murine J774A.1 cell line rather than primary macrophages or in vivo models.
  • Evident from the text: some assays have limited replication (qRT-PCR used two independent biological samples; phagocytosis/killing used two independent experiments; cytometry experiments were performed in duplicates), mutant growth phenotype data are not shown, and lactate comparison is proposed for future studies.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3389/fmicb.2015.00919

Cites

Statistics

Nothing to recompute.

Claims the paper makes

0 of 68 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    In the presence of acetic acid, biofilm biomass as well as viable cell numbers were reduced, when compared to RPMI medium only.

    “In the presence of acetic acid, biofilm biomass as well as viable cell numbers were reduced, when compared to RPMI medium only (Figure 1).”
    Quote in the paperSubstantial claim
  • Findingown data

    The percentage of phagocytosis index was significantly higher (14.7%, p < 0.001) in WT acetic acid-adapted cells compared with C. glabrata cells grown without the acid (8.1%).

    “In addition, the percentage of phagocytosis index was also significantly higher (14.7%, p < 0.001) in WT acetic acid-adapted cells in comparison with C. glabrata cells grown without the acid (8.1%; Figure 3D).”
    Quote in the paperSubstantial claim
  • Findingown data

    No significant difference in the phagocytosis index was observed for fps1 mutant cells grown in RPMI compared with WT cells.

    “Regarding fps1 mutant cells, no significant difference in the phagocytosis index was observed in cells grown in RPMI in comparison with WT cells.”
    Quote in the paperSubstantial claim
  • Findingown data

    When planktonic cells were grown in the presence of acetic acid with 50 μg/ml fluconazole, a decrease in ADY2a (p < 0.001) expression was observed.

    “However, when planktonic cells were grown in the presence of acetic acid with 50 μg/ml fluconazole, a decrease in ADY2a (p < 0.001) expression was observed (Figure 2A).”
    Quote in the paperSubstantial claim
  • Findingown data

    Upon 30 min of co-incubation, flow cytometry analysis showed that a higher percentage of macrophages with internalized yeast cells are present in comparison with the percentage of macrophages with adherent or adherent and internalized yeast cells.

    “In general, upon 30 min of co-incubation, flow cytometry analysis showed that a higher percentage of macrophages with internalized yeast cells are present in comparison with the percentage of macrophages with adherent or, adherent and internalized yeast cells (Figure 3).”
    Quote in the paperNot a substantial claim
  • May combine two claims. 33
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 47

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    For biofilm cells, the response of ADY2a, ADY2b, FPS1, FPS2, and ATO3 genes to fluconazole was dependent on the concentration of the drug: an increase with 312.5 μg/ml of fluconazole and a decrease with the highest drug concentration (1250 μg/ml); this behavior was identical for cells grown either in the presence or absence of acetic acid, except for ADY2a, which displayed either a maintenance of,

    “The drug effect was also studied for biofilm cells in the presence of either 312.5 or 1250 μg/ml fluconazole. The response of these genes was dependent on the concentration of the drug and a clear pattern of expression was found: an increase with 312.5 μg/ml of fluconazole and a decrease with the highest drug concentration (1250 μg/ml). This behavior was identical for cells grown either in the”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The amount of TNF-α was similar in all conditions.

    “The amount of this cytokine was similar in all conditions.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In the presence of acetic acid, ady2a killing was significantly lower than WT (p < 0.05) and fps1 (p < 0.001).

    “The percentage of killing did not change with longer incubation periods but was significantly lower than WT or fps1 strains, in the presence of acetic acid, for ady2a (WT vs. ady2a, p < 0.05 and fps1 vs. ady2a, p < 0.001).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In planktonic cells grown in RPMI with 50 μg/ml fluconazole, FPS1 expression increased (p < 0.05) and ADY2a expression was maintained, in comparison with media without the drug.

    “In planktonic cells grown in RPMI with 50 μg/ml fluconazole, FPS1 expression increased (p < 0.05) and ADY2a expression was maintained, in comparison with media without the drug.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    For both conditions (with and without acetic acid), a statistically significant reduction in biofilm biomass and in CFU count was observed with the highest concentration of fluconazole for mutant biofilms, similar to the WT strain; no statistically significant differences were observed between mutant and WT strains.

    “For both conditions, a statistically significant reduction in biofilm biomass and in CFU count was observed with the highest concentration of fluconazole. This was a similar result to the one found for the WT strain. Moreover, no statistically significant differences were observed between mutant and WT strains.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In RPMI medium, the MIC value for C. glabrata ATCC 2001 planktonic cells was 150 μg/mL and the MFC value was 312.5 μg/ml.

    “In RPMI medium, we obtained a MIC value of 150 μg/mL for C. glabrata cells and a minimal fungicidal concentration (MFC) value of 312.5 μg/ml (Table 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the absence of fluconazole, ADY2b, FPS1, FPS2, and ATO3 genes displayed a very weak level of expression in RPMI medium, both in planktonic and biofilm cells; however, the gene ADY2a was strongly induced in these conditions.

    “In the absence of fluconazole, ADY2b, FPS1, FPS2, and ATO3 genes displayed a very weak level of expression in RPMI medium, both in planktonic and biofilm cells. However, the gene ADY2a was strongly induced in these conditions (Figures 2A,B).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The C. glabrata genome encodes two putative homologues of ScADY2 (ADY2a and ADY2b) and one of ScATO3 (ATO3), all functionally uncharacterized.
    • Rests on an assumption: The C. glabrata homologues ADY2a, ADY2b, and ATO3 share the stated sequence identities with their S. cerevisiae counterparts.

  • May combine two claims.

    Findingown data

    For yeast cells grown in the presence of acetic acid, fps1 mutant behaved similarly to WT cells, while ady2a cells were more resistant to killing.

    “However, for yeast cells grown in the presence of acetic acid, fps1 mutant behaved similarly to WT cells, while ady2a cells were more resistant to killing.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    When cells were grown in the presence of acetic acid, all genes displayed a higher level of expression, except for FPS2, which was expressed at low levels; this increase was observed in both planktonic and biofilm cells and was very striking for the ADY2a gene (p < 0.001 and p < 0.01, respectively).

    “When cells were grown in the presence of acetic acid, all genes displayed a higher level of expression, except for FPS2, which was expressed at low levels. This increase was observed in both planktonic and biofilm cells (Figures 2A,B) and it was very striking for the ADY2a gene (p < 0.001 and p < 0.01, respectively).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The C. glabrata genome encodes two putative homologues of ScADY2 (ADY2a and ADY2b) and one of ScATO3 (ATO3), all functionally uncharacterized.
    • Rests on an assumption: The C. glabrata homologues ADY2a, ADY2b, and ATO3 share the stated sequence identities with their S. cerevisiae counterparts.

  • May combine two claims.

    Findingown data

    Both fps1 and ady2a cells appear more susceptible to macrophage killing than WT cells when grown in RPMI media, particularly at the early time point, although no statistical significance was reached.

    “Regarding C. glabrata mutant strains, although no statistical significance was reached, both fps1 and ady2a cells appear to be more susceptible to macrophage killing than WT cells, when grown in RPMI media, in particularly at the early time point (Figure 4A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In both conditions, aggregates of yeast cells were observed in the biofilms; however, in the presence of acetic acid these aggregates were smaller.

    “In both conditions, aggregates of yeast cells were observed in the biofilms. However, in the presence of acetic acid these aggregates were smaller (Figure 1C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Biofilms obtained for the ady2a and fps1 mutants and for the WT strains were similar, and all strains displayed increased biofilm biomass in media without acetic acid.

    “Notably, biofilms obtained for the mutants and for the WT strains were similar, and all strains displayed increased biofilm biomass in media without acetic acid.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    A high fluconazole concentration leads to a basal level of expression for all genes, both in the presence and absence of acetic acid, although viable cells are still found.

    “A high fluconazole concentration however leads to a basal level of expression for all genes, both in the presence and absence of acetic acid, although viable cells are still found in these conditions.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    C. albicans growth on an alternative carbon source, such as lactate, can affect stress adaptation, antifungal drug resistance, the cell wall architecture and proteome including immune detection.

    “Recently, it has been shown that C. albicans growth on an alternative carbon source, such as lactate, can affect stress adaptation, antifungal drug resistance, the cell wall architecture and proteome including immune detection (Ene et al., 2012a,b, 2013).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Ene et al., 2012a,b, 2013 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Candida glabrata is a prevalent human fungal pathogen and the second most frequent cause of candidiasis after Candida albicans in the United States.

    “Candida glabrata is a prevalent human fungal pathogen that has become the second most frequent cause of candidiasis after Candida albicans in the United States, according to the SENTRY Program (Pfaller et al., 2001, 2011).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Pfaller et al., 2001, 2011

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    C. glabrata infections are especially difficult to treat due to a high inherent antifungal resistance, particularly against azoles.

    “C. glabrata infections are especially difficult to treat due to a high inherent antifungal resistance, particularly against azoles (Pfaller, 2012).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Pfaller, 2012 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    C. glabrata colonizes host tissues as well as abiotic surfaces as biofilms.

    “Moreover, C. glabrata colonizes host tissues as well as abiotic surfaces as biofilms (Silva et al., 2009).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Silva et al., 2009 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Lactate-grown C. albicans cells were taken up by macrophages less efficiently, but they were more efficient at killing and escaping phagocytosis.

    “Lactate-grown cells were taken up by macrophages less efficiently, but they were more efficient at killing and escaping phagocytosis (Ene et al., 2013).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Ene et al., 2013 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The formation of Candida biofilms raises important clinical issues because of a significant further increase in antifungal drug resistance as well as evasion of host immune defenses.

    “The formation of Candida biofilms raises important clinical issues because of a significant further increase in antifungal drug resistance as well as evasion of host immune defenses.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Douglas, 2003 source

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    • cites Ramage et al., 2005 source

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    • cites Calderone and Clancy, 2012

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Besides Ady2, Fps1 is a channel promoting the facilitated diffusion of undissociated acetic acid (CH3COOH) at low pH in S. cerevisiae.

    “Besides Ady2, Fps1 is a channel promoting the facilitated diffusion of undissociated acetic acid (CH3COOH) at low pH in S. cerevisiae (Mollapour and Piper, 2007).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Mollapour and Piper, 2007

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  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Macrophages without yeast cells supernatants were used as a negative control, and macrophages infected with Escherichia coli cells were used as a positive control (LPS control).

    “Macrophages without yeast cells supernatants were used as a negative control, and macrophages infected with Escherichia coli cells were used as a positive control (LPS control).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    MIC assays were performed by the microdilution method according to CLSI document M27-A3, with some modifications.

    “The MIC assays were performed by the microdilution method according to the Clinical and Laboratory Standards Institute (CLSI, 2008) document M27-A3, with some modifications, using RPMI 1640 broth, supplemented with 0.165 M of MOPS, with or without adding 0.5% acetic acid at pH 5.0 (Pfaller et al., 2008).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites CLSI, 2008

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In S. cerevisiae, ADY2 (also called ATO1) encodes a permease-mediating uptake of acetate-propionate-formate present in ethanol or acetate-grown cells.

    “In S. cerevisiae, ADY2 (also called ATO1) encodes a permease-mediating uptake of acetate–propionate–formate present in ethanol or acetate-grown cells (Casal et al., 1996; Paiva et al., 2004; Pacheco et al., 2012).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Casal et al., 1996 source

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    • cites Paiva et al., 2004 source

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    • cites Pacheco et al., 2012 source

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    Premisecitation

    This hypothesis was supported by the apparent expression regulation of two C. albicans carboxylic acids transporter genes, JEN1 and JEN2, following macrophages internalization.

    “This hypothesis was supported by the apparent expression regulation of two C. albicans carboxylic acids transporter genes, JEN1 and JEN2, following macrophages internalization.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Soares-Silva et al., 2004 source

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    • cites Vieira et al., 2010 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    At least two C. glabrata homologues of ScFps1 have been described: CAGL0C03267g (designated FPS1) and CAGL0E03894g (designated FPS2).

    “At least two C. glabrata homologues of ScFps1 have been described: CAGL0C03267g (designated FPS1) and CAGL0E03894g (designated FPS2; Beese-Sims et al., 2012).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Beese-Sims et al., 2012 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Biofilm experiments were performed at least three times using independent biological samples.

    “Experiments were performed at least three times using independent biological samples.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    C. glabrata cells resist killing by macrophages and have evolved distinct strategies for intracellular survival.

    “Of note, C. glabrata cells also resist killing by macrophages and have evolved distinct strategies for intracellular survival (Otto and Howard, 1976; Kaur et al., 2007; Seider et al., 2010).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Otto and Howard, 1976

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    • cites Kaur et al., 2007 source

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    • cites Seider et al., 2010 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In C. glabrata, lactate assimilation is at least required for survival in the intestine.

    “In C. glabrata, lactate assimilation is at least required for the survival in the intestine (Ueno et al., 2011).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Ueno et al., 2011 source

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  • May combine two claims.

    Premisecitation

    A transcription profile of C. glabrata following macrophage internalization revealed the up-regulation of genes involved in gluconeogenesis, β-oxidation and glyoxylate cycle, similar to what has been described for C. albicans; moreover, a putative acetate transporter (ADY2a) appears to be induced.

    “A transcription profile of C. glabrata following macrophage internalization revealed the up-regulation of genes involved in gluconeogenesis, β-oxidation and glyoxylate cycle, similar to what has been described for C. albicans. Moreover, a putative acetate transporter (ADY2a) appears to be induced (Kaur et al., 2007).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Kaur et al., 2007 source

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  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    The induction of ADY2a was also observed when C. glabrata is facing neutrophils, implying that in this environment, C. glabrata may assimilate acetate and other short-chain carboxylic acids for use as an alternative carbon source.

    “The induction of ADY2a was also observed when C. glabrata is facing neutrophils (Fukuda et al., 2013), implying that in this environment, C. glabrata may assimilate acetate and other short-chain carboxylic acids for use as an alternative carbon source.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Fukuda et al., 2013 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Biofilm viability experiments were performed in duplicate and at least three times using independent biological samples.

    “Experiments were performed in duplicate and at least three times using independent biological samples.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    In C. albicans, evidence exists that growth in the presence of an alternative carbon source affects phagocytosis.

    “In the case of C. albicans, evidence exists that growth in the presence of an alternative carbon sources affects phagocytosis (Ene et al., 2013).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Ene et al., 2013 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Gene expression experiments were performed in two independent biological samples.

    “Experiments were performed in two independent biological samples.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Controls consisting of yeast cells grown in the same conditions without macrophages were added.

    “Controls consisting of yeast cells grown in the same conditions without macrophages were added.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    A negative control without template was conducted for each gene in each PCR run, and a control for DNA contamination was implemented by using the purified RNA samples as template.

    “A negative control without template was conducted for each gene in each PCR run, and a control for DNA contamination was implemented by using the purified RNA samples as template.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All the experiments were performed in triplicate, at least in three independent assays.

    “All the experiments were performed in triplicate, at least in three independent assays.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    When C. albicans cells face a glucose-poor niche their metabolism changes; after phagocytosis by macrophages, repression of glycolysis occurs, and the glyoxylate cycle, gluconeogenesis, and β-oxidation are induced.

    “When C. albicans cells face a glucose-poor niche their metabolism changes. For instance, after phagocytosis of C. albicans by macrophages, repression of glycolysis occurs, the glyoxylate cycle, gluconeogenesis, and β-oxidation (Fradin et al., 2003, 2005; Prigneau et al., 2003; Lorenz et al., 2004; Barelle et al., 2006; Wilson et al., 2009) are induced.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Fradin et al., 2003, 2005

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Prigneau et al., 2003 source

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    • cites Lorenz et al., 2004 source

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    Premisecitation

    It was postulated that acetyl-CoA feeding into the glyoxylate cycle might be derived from other non-fermentable carbon sources, such as lactate or acetate.

    “Thereby, it was postulated that acetyl-CoA feeding into the glyoxylate cycle might be derived from other non-fermentable carbon sources, such as lactate or acetate (Piekarska et al., 2006; Ramirez and Lorenz, 2007).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Piekarska et al., 2006 source

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    • cites Ramirez and Lorenz, 2007 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    These genes may act as glycerol channels, as the double mutant fps1 fps2 accumulates glycerol, displays constitutive cell wall stress and hypersensitivity to treatment by caspofungin.

    “These genes may act as glycerol channels, as the double mutant fps1 fps2 accumulates glycerol, displays constitutive cell wall stress and hypersensitivity to treatment by caspofungin (Beese-Sims et al., 2012).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Beese-Sims et al., 2012 source

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  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Data obtained with mutant strains in these pathways using mouse models of systemic candidiasis showed that the glyoxylate cycle is required for virulence, while β-oxidation is not.

    “Additionally, data obtained with mutant strains in these pathways using mouse models of systemic candidiasis showed that the glyoxylate cycle is required for virulence (Lorenz and Fink, 2001, 2002; Barelle et al., 2006; Han et al., 2011), while β-oxidation is not (Piekarska et al., 2006).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Lorenz and Fink, 2001, 2002

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Barelle et al., 2006 source

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    • cites Han et al., 2011 source

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    Premisecitation

    In conjunction with its two additional homologues, ATO2 and ATO3, ADY2 has also been implicated in ammonia export.

    “In conjunction with its two additional homologues, ATO2 and ATO3, it has also been implicated in ammonia export (Palkova et al., 2002; Vachova et al., 2009).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Palkova et al., 2002 source

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    • cites Vachova et al., 2009 source

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    Study design and conductown data

    Cytokine quantification experiments were performed in triplicate.

    “These experiments were performed in triplicate.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Phagocytosis assays were performed in two independent experiments.

    “Two independent experiments of this assay were performed.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Acetic acid is present in other human niches, including the vaginal mucosa and vaginal secretions.

    “Moreover, this carboxylic acid is also present in other human niches, including the vaginal mucosa (Preti et al., 1979) and vaginal secretions (Huggins and Preti, 1976).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Preti et al., 1979 source

      Not checked in the free run.

    • cites Huggins and Preti, 1976

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    Differences in phagocytosis may reflect alterations in the cell surface where PAMPs are present, given the major impact of carbon source on C. albicans cell surface.

    “Given the major impact of carbon source on C. albicans cell surface observed in some studies (Ene et al., 2012a,b), the differences reported in terms of phagocytosis may reflect these alterations in cells surface, where PAMPs are present.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Ene et al., 2012a,b

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Cyb2 is required for the assimilation of lactate, a non-fermentable carbon source present in the intestine.

    “This protein is required for the assimilation of lactate, a non-fermentable carbon source present in the intestine (Ueno et al., 2011).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Ueno et al., 2011 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Fluconazole was fungicidal for C. albicans in a synthetic vagina-simulative medium with an acidic pH.

    “In fact, Moosa et al. (2004) showed that fluconazole was fungicidal for C. albicans in a synthetic vagina-simulative medium with an acidic pH.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Moosa et al., 2004 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In the intestine, where glucose is scarce, C. glabrata requires Cyb2 for adaptation and survival.

    “In the intestine, where glucose is scarce, C. glabrata requires Cyb2 for adaptation and survival (Ueno et al., 2011).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Ueno et al., 2011 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Lactate-grown C. albicans cells were more efficient at killing and escaping from macrophages.

    “This work reported that lactate grown C. albicans cells were more efficient at killing and escaping from macrophages (Ene et al., 2013).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Ene et al., 2013 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Acetic acid acted synergistically with clotrimazole and flucytosine in C. glabrata.

    “Another study reported that acetic acid, other alternative carbon source, acted synergistically with clotrimazole and flucytosine in C. glabrata (Costa et al., 2013).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Costa et al., 2013 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    fps1 was more susceptible to caspofungin, an antifungal agent that targets cell wall glucans.

    “Beese-Sims et al. (2012) demonstrated that fps1 was more susceptible to caspofungin, an antifungal agent that targets cell wall glucans.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Beese-Sims et al., 2012 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Inside phagosomes, C. glabrata faces glucose-limited environments and must change its metabolism to utilize non-fermentable carbon sources.

    “In such niches, for example inside phagosomes, it faces glucose-limited environments. To survive, it has to change its metabolism and utilize non-fermentable carbon sources (Kaur et al., 2007; Roetzer et al., 2010; Fukuda et al., 2013).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Kaur et al., 2007 source

      Not checked in the free run.

    • cites Roetzer et al., 2010 source

      Not checked in the free run.

    • cites Fukuda et al., 2013 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Acetate is a weak organic acid present in the intestine and can be produced by intestinal microbiota.

    “Other weak organic acids besides lactate are present in the intestine, namely acetate, which can be produced by intestinal microbiota (Yamaguchi et al., 2005).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Yamaguchi et al., 2005 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Acetic acid was responsible for the synergistic, fungicidal effect in that medium.

    “The analysis of the components of the medium indicated that acetic acid was responsible for the synergistic, fungicidal effect (Moosa et al., 2004).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Moosa et al., 2004 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    C. albicans grown in the presence of lactic acid can resist phagocytosis better than when grown in glucose.

    “Recently, it has been described that C. albicans grown in the presence of lactic acid can resist to phagocytosis better than when grown in glucose (Ene et al., 2013).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Ene et al., 2013 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Many receptors recognize components of the fungal cell wall leading to phagocytosis of the fungal cell.

    “Many receptors recognize components of the fungal cell wall leading to the phagocytosis of the fungal cell (Netea and Marodi, 2010; Gow et al., 2012).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Netea and Marodi, 2010

      Not checked in the free run.

    • cites Gow et al., 2012 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    C. albicans cells grown in lactic acid were more resistant to amphotericin B but more susceptible to miconazole.

    “Ene et al. (2012a) showed that C. albicans cells grown in an alternative carbon source, lactic acid, were more resistant to amphotericin B but more susceptible to miconazole.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Ene et al., 2012a

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Acetic acid influences C. glabrata behavior in biofilm formation, antifungal drug resistance and phagocytosis.

    “This study shows that acetic acid influences C. glabrata behavior in biofilm formation, antifungal drug resistance and phagocytosis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Ady2a may play a role in phagocytosis, and carbon source clearly affects the cell response to macrophage killing.

    “This suggests a potential role of Ady2a in phagocytosis and reveals that the carbon source clearly affects the cells response to macrophage killing.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The mutant strains ady2a and fps1 are functional nulls with no secondary mutations affecting the phenotypes.

  • May combine two claims.

    Interpretationown data

    WT C. glabrata cells grown in the presence of acetic acid are better internalized and killed by macrophages.

    “Taken together, these results show that WT C. glabrata cells, grown in the presence of acetic acid, are better internalized and killed by macrophages and suggest that acetic acid renders ady2a mutant more resistance to macrophage killing.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The glyoxylate cycle is significant in the virulence of C. albicans and C. glabrata, and ADY2 and FPS1 are expressed inside macrophages.

    “As previously mentioned, some studies have recognized the significance of the glyoxylate cycle in the virulence of C. albicans (Ramirez and Lorenz, 2007) and C. glabrata (Kaur et al., 2007; Fukuda et al., 2013) and have shown that ADY2 and FPS1 are expressed inside macrophages (Kaur et al., 2007; Seider et al., 2011).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Ramirez and Lorenz, 2007 source

      Not checked in the free run.

    • cites Kaur et al., 2007 source

      Not checked in the free run.

    • cites Fukuda et al., 2013 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Previous studies reported ADY2a upregulation in C. glabrata cells upon macrophage phagocytosis.

    “Of note, previous studies reported an ADY2a upregulation in C. glabrata cells upon macrophage phagocytosis (Kaur et al., 2007; Seider et al., 2011).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Kaur et al., 2007 source

      Not checked in the free run.

    • cites Seider et al., 2011 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Biofilms needed about 10 to 100 times more antifungal drug to be eradicated compared with planktonic cells.

    “These results are consistent with reports for C. albicans biofilms (Baillie and Douglas, 1998; Chandra et al., 2001a,b; Ramage et al., 2001), showing that biofilms needed about 10 to 100 times more quantity of antifungal drug to be eradicated when compared to planktonic cells.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Baillie and Douglas, 1998

      Not checked in the free run.

    • cites Chandra et al., 2001a,b

      Not checked in the free run.

    • cites Ramage et al., 2001 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The finding that acetic acid increases fluconazole susceptibility has important clinical impact in the treatment of vaginal candidiasis because this niche has an acidic pH and is rich in alternative carbon sources.

    “This finding has an important clinical impact, namely in the treatment of vaginal candidiasis, since this niche has an acidic pH and is rich in alternative carbon sources, such as lactic and acetic acid.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In S. cerevisiae, acetic acid can serve as an alternative carbon source to fuel gluconeogenesis via the glyoxylate cycle.

    “In S. cerevisiae acetic acid can serve as an alternative carbon source to fuel gluconeogenesis via the glyoxylate cycle (Turcotte et al., 2010).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Turcotte et al., 2010 source

      Not checked in the free run.

65 of 65

  • The paper relies on this without support.

    Vaginal candidiasis involves acidic pH niches containing acetic acid.

    “These results highlight how acidic pH niches, associated with the presence of acetic acid, can impact in the treatment of C. glabrata infections, in particular in vaginal candidiasis.”

  • The paper relies on this without support.

    The C. glabrata genome encodes two putative homologues of ScADY2 (ADY2a and ADY2b) and one of ScATO3 (ATO3), all functionally uncharacterized.

    “The C. glabrata genome encodes two putative homologues of ScADY2 and one of ScATO3, all of which remain functionally uncharacterized.”

  • The paper relies on this without support.

    The C. glabrata homologues ADY2a, ADY2b, and ATO3 share the stated sequence identities with their S. cerevisiae counterparts.

    “These C. glabrata homologues are CAGL0M03465g (designated ADY2a) and CAGL0L07766g (designated ADY2b), sharing 74 and 73% identity with ScADY2, respectively, and CAGL0A03212g (designated ATO3), sharing 62% identity with ScATO3.”

  • The paper relies on this without support.

    The in vitro macrophage phagocytosis and killing assays reflect the interaction of C. glabrata with macrophages in human host niches.

    “To evaluate the effect of acetic acid adaptation on phagocyte interaction, macrophages were infected with C. glabrata, previously grown in the presence or absence of acetic acid, and yeast killing was assessed by CFU after 3 and 18 h of co-incubation.”

  • The paper relies on this without support.

    The mutant strains ady2a and fps1 are functional nulls with no secondary mutations affecting the phenotypes.

    “We also assessed the capacity of macrophages to phagocytose C. glabrata ady2a and fps1 mutant cells pre-grown in RPMI with or without acetic acid.”
5 of 5

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Lactate-grown C. glabrata cells resist killing by macrophages and have developed distinct tactics for intracellular survival and escaping phagocytosis.

    “Lactate-grown C. glabrata cells, for example, resist killing by macrophages and have developed distinct tactics for intracellular survival killing and escaping phagocytosis [41].”

    From Candida glabrata: pathogenicity and resistance mechanisms for adaptation and survival (2021) · cites this paper as [41]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Biofilm fluconazole susceptibility is described in opposite directions

    Minor

    The heading says biofilms are more susceptible than planktonic cells, but the results report that biofilms show a significant reduction only at 1250 μg/ml and are not eradicated, whereas Table 3 gives planktonic-cell MICs of 150 and 50 μg/ml.

  • FPS2 is both listed as upregulated and excluded from that result

    Minor

    The abstract includes FPS2 among genes upregulated in the presence of acetic acid, while the results explicitly identify FPS2 as the exception to the higher-expression pattern.

2 of 2

The citation lists haven’t been fetched yet

References 68 · Cited by 49

Research works printed but not linked: 1

  1. Pfaller, 2008, Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts:Approved Standard

Authors
Mota, S., Alves, R., Carneiro, C., Silva, S., Brown, A. J., Istel, F., Kuchler, K., Sampaio, P., Casal, M., Henriques, M., & Paiva, S.
Affiliations
  • Universidade Técnica do Porto (PT)
  • University of Minho (PT)
  • University of Aberdeen (GB)
  • Max Perutz Labs (AT)
Corresponding author
Paiva, Sandra
ORCID
Venue
Frontiers in Microbiology
Publisher
Frontiers Media SA
Published
2015-09-04
Volume
6
Pages
919-919
ISSN
1664-302X (ISSN-L)
DOI
10.3389/fmicb.2015.00919
PMC
PMC4560035
OpenAlex
W1821298691
Semantic Scholar
91023c87c7aba87271021470dd48e42b357c7d2f
Cited by
49
References
68
Citations by year
2026: 2 · 2025: 4 · 2024: 4 · 2023: 4 · 2022: 7 · 2021: 10 · 2020: 5 · 2019: 5 · 2018: 2 · 2017: 3 · 2016: 2 · 2015: 1
FWCI
2.07
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Frontiers in Microbiology (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Aberdeen University Research Archive (Aberdeen University) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Portuguese National Funding Agency for Science, Research and Technology (RCAAP Project by FCT) (submittedVersion, other-oa)
Funders
  • Pfizer
  • China Building Materials Academy (PEst-OE/BIA/UI4050/2014)
  • Programa Operacional Temático Factores de Competitividade (PEst-OE/BIA/UI4050/2014, PEst-OE/EQB/LA0023/2013, PTDC/SAU-MIC/119069/2010, UID/BIA/04050/2013)
  • Fundação para a Ciência e a Tecnologia (COMPETE, PTDC/SAU-MIC/119069/2010, PEst-OE/BIA/UI4050/2014, UID/BIA/04050/2013, PEst-OE/EQB/LA0023/2013)
Topics
  • Health Sciences › Medicine › Infectious Diseases › Antifungal resistance and susceptibility
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
  • Health Sciences › Medicine › Epidemiology › Fungal Infections and Studies
Keywords
Candida glabrata, acetic acid, alternative carbon sources, antifungal susceptibility, fluconazole resistance, phagocytosis, macrophages, biofilm formation, nutrient deprivation, FPS1, vaginal candidiasis, opportunistic fungal infection, low pH

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-09.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.

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