Candida glabrata : new tools and technologies—expanding the toolkit

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Candida glabrata belongs to the non-CTG clade, unlike most well-studied Candida species.

“Phylogenetically, it is more closely related to Saccharomyces cerevisiae than to any other Candida species, see Fig. 1, and unlike most well-studied Candida species, it belongs to the non-CTG clade species (Dujon et al. 2004; Ahmad et al. 2014).”

• cites Dujon et al. 2004Not checked yet

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• cites Ahmad et al. 2014Not checked yet

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Candida glabrata is associated with longer hospital stays and higher costs than Candida albicans.

“Of interest, in the rise of non-albicans Candida species, is C. glabrata which has been shown to be associated with longer hospital stays and higher costs than C. albicans (Moran et al. 2010).”

• cites Moran et al. 2010Not checked yet

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Candida glabrata is phylogenetically more closely related to Saccharomyces cerevisiae than to any other Candida species.

“Phylogenetically, it is more closely related to Saccharomyces cerevisiae than to any other Candida species, see Fig. 1, and unlike most well-studied Candida species, it belongs to the non-CTG clade species (Dujon et al. 2004; Ahmad et al. 2014).”

• cites Dujon et al. 2004Not checked yet

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• cites Ahmad et al. 2014Not checked yet

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Candida glabrata has no known documented sexual cycle.

“Furthermore, C. glabrata has no known documented sexual cycle although both mating types are commonly found and its genome contains homologues of the majority of the genes involved in mating in S. cerevisiae (Muller et al. 2008).”

• cites Muller et al. 2008Not checked yet

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Saccharomyces cerevisiae has one of the most diverse plasmid toolkits available for ease of investigating and manipulating genes.

“Unsurprisingly, the model organism, S. cerevisiae, has one of the most diverse plasmid toolkits available for ease of investigating and manipulating genes, from expression plasmids to reporter plasmids.”

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Aoyama et al. (2014) used RNA-seq to investigate transcriptional initiation in Candida glabrata and predicted 50 new Candida glabrata genes.

“for example, Aoyama et al. (2014) used RNA-seq to investigate transcriptional initiation in C. glabrata and predicted 50 new C. glabrata genes.”

• cites Aoyama et al. 2014 sourceNot checked yet

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One likely reason the Candida glabrata community has not widely used the reversible YKU80 disruption strain is because target efficiency is only increased by 5.1% and disruption of YKU80 leads to synthetic sickness or lethality in Saccharomyces cerevisiae.

“One likely reason that the C. glabrata community has not widely used the reversible YKU80 disruption strain for generating knockouts is because the target efficiency is only increased by 5.1% and disruption of YKU80 leads to synthetic sickness or lethality in S. cerevisiae (Ueno et al. 2007); furthermore, deletions of YKU70/80 in C. glabrata have been shown to affect subtelomeric silencing (Rosas-”

• cites Ueno et al. 2007Not checked yet

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Although the Candida glabrata deletion collection currently comprises only 12% of the genome, advances have already been made in understanding the virulence mechanisms of Candida glabrata.

“Although the C. glabrata deletion collection currently comprises only 12% of the genome, advances have already been made in understanding the virulence mechanisms of C. glabrata and additional strains have been constructed (K. Haynes et al., unpublished).”

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A partial C. glabrata Gateway ORFeome has been used to examine conservation of PPIs between C. glabrata and S. cerevisiae and to perform a pooled overexpression screen to identify novel C. glabrata genes important for survival under stress conditions.

“Indeed, a partial C. glabrata Gateway ORFeome has been used to examine conservation of PPIs between C. glabrata and S. cerevisiae (Ho and Huvet et al., unpublished) and to perform a pooled overexpression screen to identify novel C. glabrata genes important for survival under stress conditions (Ho et al., unpublished).”

• cites Ho and Huvet et al., unpublishedNot checked yet

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• cites Ho et al., unpublishedNot checked yet

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Gene targeting can be performed more efficiently in the newly constructed lig4 strain compared to the yku70/80 strains and does not appear to cause any phenotype upon its deletion.

“As gene targeting can be performed more efficiently in this newly constructed lig4 strain compared to the yku70/80 strains and does not appear to cause any phenotype upon its deletion, it will be interesting to see if the Candida community will adopt this strain for use in high-throughput knockout generation in the near future.”

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The construction of a Candida glabrata Gateway adapted ORFeome would alleviate the need to prioritize genes individually, as any gene of interest could easily be shuttled into a range of different destination vectors.

“The construction of a C. glabrata Gateway adapted ORFeome would alleviate this, as any gene of interest could easily be shuttled into a range of different destination vectors thereby”

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Once an Entry clone is available, it is possible to shuttle the gene of interest to any available Gateway adapted vector; furthermore, this process is bidirectional.

“However, once an Entry clone is available, it is possible to shuttle the gene of interest to any available Gateway adapted vector; furthermore, this process is bidirectional (Hartley 2000)”

• cites Hartley 2000Not checked yet

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Cen Fiori and Dijck (2015) constructed a lig4 mutant and showed that gene targeting efficiency increased up to 35 times compared to the parental strain when using 40-bp flanking homologous DNA fragments.

“More recently, Cen Fiori and Dijck (2015) have constructed a lig4 mutant (Lig4 is a DNA ligase involved in NHEJ) and shown that gene targeting efficiency increased up to 35 times compared to the parental strain when using 40-bp flanking homologous DNA fragments.”

• cites Cen, Fiori and Dijck 2015Not checked yet

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Gateway ORFeomes and compatible vectors have been created in other species including Drosophila melanogaster, Caenorhabditis elegans and Candida albicans.

“Furthermore, Gateway ORFeomes and compatible vectors have been created in other species including D. melanogaster, Caenorhabditis elegans and C. albicans (Reboul et al. 2003; Akbari et al. 2009; Chauvel et al. 2012)”

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• cites Akbari et al. 2009Not checked yet

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• cites Chauvel et al. 2012Not checked yet

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The Gateway® Technology facilitates high-efficiency transfer of genes between different Gateway vectors by site-specific recombination.

“This toolkit has recently been expanded with the introduction of the Gateway® Technology which facilitates high-efficiency transfer of genes between different Gateway vectors by site-specific recombination (Hartley 2000).”

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A drawback to the Gateway system is that it requires the initial insertion of the gene of interest into a plasmid with the two flanking Gateway recombination sites (attL1 and attL2) to produce an initial Gateway Entry clone.

“A drawback to the system is that it requires the initial insertion of the gene of interest into a plasmid with the two flanking Gateway recombination sites (attL1 and attL2) to produce an initial Gateway Entry clone (Hartley 2000).”

• cites Hartley 2000Not checked yet

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Compared with Saccharomyces cerevisiae, Candida glabrata has been less well studied and has fewer molecular tools and resources available to interrogate its biology.

“Compared with the model organism S. cerevisiae, C. glabrata has been less well studied, and as such there are fewer molecular tools and resources available to interrogate its biology; see Table 1 for a summary.”

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Until recently, no deletion collection existed for Candida glabrata; thus, research has focused on Candida glabrata genes known to impact virulence in other pathogens, or on orthologues of Saccharomyces cerevisiae genes.

“Until recently, no deletion collection existed for C. glabrata; thus, research has focused on C. glabrata genes known to impact virulence in other pathogens, or on orthologues of S. cerevisiae genes that may or may not encode functions important for infection.”

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The Candida glabrata deletion collection currently comprises 619 strains representing approximately 12% of the predicted genome.

“The C. glabrata deletion collection currently comprises 619 strains representing approximately 12% of the predicted genome (Schwarzmüller et al. 2014).”

• cites Schwarzmüller et al. 2014Not checked yet

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Although a full Candida glabrata ORFeome is yet to be published, the Gateway system is already being utilized by Candida glabrata researchers to investigate genes of interest.

“Although a full C. glabrata ORFeome is yet to be published, the Gateway system is already being utilized by C. glabrata researchers to investigate genes of interest.”

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The Saccharomyces cerevisiae Yeast Knockout Collection comprises 5916 individual genes knocked out in one or more of four backgrounds.

“Since the release of the S. cerevisiae Yeast Knockout Collections (YKO), which comprises 5916 individual genes knocked out in one or more of four backgrounds (MATa, MATα, heterozygous diploid, and homozygous diploid) (Winzeler et al. 1999; Giaever et al. 2002)”

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• cites Giaever et al. 2002Not checked yet

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Loss of HIS3, LEU2, TRP1 or a combination of all three in Candida glabrata does not impact its virulence, unlike deletion of the URA3 marker in Candida albicans which is known to affect its virulence.

“Pertinently, Jacobsen et al. have shown that loss of HIS3, LEU2, TRP1 or a combination of all three in C. glabrata does not impact its virulence, unlike deletion of the URA3 marker in C. albicans which is known to affect its virulence (Lay et al. 1998; Brand et al. 2004; Jacobsen et al. 2010).”

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• cites Brand et al. 2004Not checked yet

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• cites Jacobsen et al. 2010Not checked yet

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Schwarzmüller et al. (2014) identified 28 knockout strains that are hypersensitive to caspofungin and have not been described in Saccharomyces cerevisiae nor Candida albicans, implying that Candida glabrata may have unknown mechanisms of caspofungin resistance.

“Furthermore, they identified 28 knockout strains that are hypersensitive to caspofungin and have not been described in S. cerevisiae nor C. albicans, implying that C. glabrata may have unknown mechanisms of caspofungin resistance (Schwarzmüller et al. 2014).”

• cites Schwarzmüller et al. 2014Not checked yet

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Ueno et al. generated a reversible YKU80 disruption strain that increased gene targeting efficiency by 5.1% using 40-bp flanking homologous DNA, but this strain has not gained widespread use from the community.

“Although attempts have been made to overcome this problem in C. glabrata, for example, Ueno et al. (2007) generated a reversible YKU80 disruption strain (Yku80 is involved in the NHEJ pathway) thereby increasing gene targeting efficiency by 5.1% using 40-bp flanking homologous DNA, this strain has not gained widespread use from the community.”

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Many high-throughput screens using the Saccharomyces cerevisiae YKO library have produced a wealth of information that has led to functions being assigned to previously unannotated proteins.

“many high-throughput screens have been undertaken using these libraries producing a wealth of information that has led to functions being assigned to previously unannotated proteins.”

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These types of screens and analyses can easily be extended to any organism in which a deletion collection has been constructed.

“These types of screens and analyses can easily be extended to any organism in which a deletion collection has been constructed.”

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Gelperin et al. constructed the MORF library, a library containing 5854 Saccharomyces cerevisiae ORFs as C-terminal His6-HA fusions under regulated control in Gateway compatible expression plasmids.

“Utilizing Gateway Technology, Gelperin et al. constructed the MORF (Moveable ORF) library, a library containing 5854 S. cerevisiae ORFs as C-terminal His6-HA fusions under regulated control in Gateway compatible expression plasmids.”

• cites Gelperin et al. 2005Not checked yet

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Using the MORF collection, Gelperin et al. performed the first systematic screen for glycosylated proteins in Saccharomyces cerevisiae and identified 454 new candidate glycoproteins.

“Using this collection, they performed the first systematic screen for glycosylated proteins in S. cerevisiae and identified 454 new candidate glycoproteins (Gelperin et al. 2005).”

• cites Gelperin et al. 2005Not checked yet

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Zebrafish were chosen as a virulence model because they have an adaptive component to their immune system, a high reproductive rate, low maintenance costs, transparent embryos that make in vivo visualization possible, and can be manipulated with a comprehensive series of molecular tools.

“Zebrafish were chosen as they have an adaptive component to their immune system, a high reproductive rate, low maintenance costs, have transparent embryos that makes in vivo visualization possible and can be manipulated with a comprehensive series of molecular tools (Chao et al. 2010).”

• cites Chao et al. 2010Not checked yet

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Toll-deficient D. melanogaster do succumb to C. glabrata infection and attenuated C. glabrata null strains can be identified using Toll-deficient flies.

“Furthermore, Toll-deficient D. melanogaster do succumb to C. glabrata infection and attenuated C. glabrata null strains can be identified using Toll-deficient flies; for example Quintin et al. observed that Toll-deficient flies injected with a double null C. glabrata yps1 yps7 (Yps1 and Yps 7 have previously been shown to be required for cell wall integrity and survival inside macrophages) succumb”

• cites Kaur, Ma and Cormack 2007Not checked yet

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• cites Quintin et al. 2013Not checked yet

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Bates et al. have preliminary data that indicate that an inoculum of 5 × 10^5-1 × 10^6 cells of C. glabrata ATCC 2001 was sufficient to observe approximately 80% mortality in G. mellonella 24 h post-infection at 37°C and that it follows a clear dose response, similar to that seen in a mouse model of dose-dependent killing.

“In contrast, Bates et al. have preliminary data that indicate that an inoculum of 5 × 105–1 × 106 cells of C. glabrata ATCC 2001 was sufficient to observe approximately 80% mortality in G. mellonella 24 h post-infection at 37°C and that it follows a clear dose response, similar to that seen in a mouse model of dose-dependent killing (Steven Bates, Exeter University, pers. Comm.).”

• cites Steven Bates, Exeter University, pers. Comm.Not checked yet

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Rocha et al. (2001) have previously shown that mice infected with C. albicans cdc35/cdc35 in the IV model of candidosis had 100% survival 42 days post-infection, while complete mortality was observed in mice infected with a C. albicans cdc35/cdc35 reconstituted with a plasmid expressing CDC35 16 days post-infection.

“Similarly, Rocha et al. (2001) have previously shown that mice infected with C. albicans cdc35/cdc35 in the IV model of candidosis had 100% survival 42 days post-infection, while complete mortality was observed in mice infected with a C. albicans cdc35/cdc35 reconstituted with a plasmid expressing CDC35 16 days post-infection.”

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Controversy remains over whether wild-type D. melanogaster is susceptible to fungal infections.

“However, controversy remains over whether wild-type D. melanogaster is susceptible to fungal infections, Alarco et al. (2004) reported that on average 85% of their wild-type D. melanogaster survived when injected with approximately 1 × 103 C. albicans cells per fly leading them to conclude that wild-type D. melanogaster is highly tolerant to fungal infection.”

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An invertebrate host may not appear relevant to human diseases, but they possess an innate immune system that shares many similarities with the innate immune response of mammals.

“Although, an invertebrate host may not appear relevant to human diseases, they possess an innate immune system that shares many similarities with the innate immune response of mammals (Kavanagh and Reeves 2004).”

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Becker et al. (2010) assayed the virulence of 177 C. albicans strains in a mouse model and identified 102 genes that play a role in host survival and establishment of an infection, including genes that encode known antifungal drug targets such as FKS1 and ERG1.

“For example, Becker et al. (2010) assayed the virulence of 177 C. albicans strains in a mouse model and identified 102 genes that play a role in host survival and establishment of an infection, including genes that encode known antifungal drug targets such as FKS1 and ERG1, validating the results of the screen for discovering potential novel drug targets.”

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A significant drawback to using an insect host is that they do not have an adaptive immune system, and integration of the innate and adaptive immune system occurs in C. albicans infections.

“However, a significant drawback to using an insect host is that they do not have an adaptive immune system, and it has been documented that, at least in the case of C. albicans infections, integration of the innate and adaptive immune system occurs, for instance, phagocytic activity is reinforced by Th1 cytokines and impaired by Th2 cytokines (Romani, Bistoni and Puccetti 1997; Romani 2000).”

• cites Romani, Bistoni and Puccetti 1997Not checked yet

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• cites Romani 2000 sourceNot checked yet

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Both mouse models enable host-pathogen interactions to be explored in vivo and allow comparison of the ability of mutants to progress disease via measurement of organ fungal burden.

“While both mouse models enable host–pathogen interactions to be explored in vivo and allow comparison of the ability of mutants to progress disease via measurement of organ fungal burden, they come with caveats.”

• cites Szabo and MacCallum (2011)Not checked yet

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Brennan et al. used the G. mellonella infection model to distinguish between avirulent, virulent and reduced virulence C. albicans strains; C. albicans hst7/hst7 was as virulent as the wild type (100% mortality at 48 h), while cpp1/cpp1 mutants were reduced in virulence compared to the wild type (90% mortality at 48 h).

“Furthermore, Brennan et al. used the G. mellonella infection model to distinguish between avirulent, virulent and reduced virulence C. albicans strains. They observed that C. albicans hst7/hst7 was as virulent as the wild type (100% mortality at 48 h), while cpp1/cpp1 mutants were reduced in virulence compared to the wild type (90% mortality at 48 h) (Brennan et al. 2002).”

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Thus, G. mellonella could develop as an important host for examining C. glabrata virulence as at a physiologically relevant temperature of 37°C it is susceptible to C. glabrata infection, but not at 30°C.

“Thus, G. mellonella could develop as an important host for examining C. glabrata virulence as at a physiologically relevant temperature of 37°C it is susceptible to C. glabrata infection, but not at 30°C.”

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Brunke et al. screened 416 C. glabrata mutants using Toll-deficient flies and showed that virulence in D. melanogaster was at least partially predictive of fitness in mice.

“Brunke et al. have combined the use of Toll-deficient flies and the deletion collection to screen 416 C. glabrata mutants to investigate whether results from this model of virulence can be used to predict the outcome of infections in murine models. They showed that virulence in D. melanogaster was at least partially predictive of fitness in mice.”

• cites Brunke et al. 2015Not checked yet

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Both Toll and Spätzle-deficient D. melanogaster are susceptible to fungal infections.

“Despite the controversy surrounding the use of wild-type D. melanogaster as a virulence model for fungal infections, both Toll and Spätzle-deficient D. melanogaster are susceptible to fungal infections (Alarco et al. 2004; Quintin et al. 2013; Panayidou, Ioannidou and Apidianakis 2014).”

• cites Alarco et al. 2004Not checked yet

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• cites Quintin et al. 2013Not checked yet

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• cites Panayidou, Ioannidou and Apidianakis 2014Not checked yet

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This correlates with previous mouse data where 50% of the mice survived 35 days post-infection with C. albicans cpp1/cpp1, while 100% mouse mortality was observed 6 days post-infection with C. albicans hst7/hst7.

“Again, this correlates with previous mouse data where 50% of the mice survived 35 days post-infection with C. albicans cpp1/cpp1 (Csank et al. 1997), while 100% mouse mortality was observed 6 days post-infection with C. albicans hst7/hst7 (Leberer et al. 1996).”

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• cites Leberer et al. 1996Not checked yet

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In the case of C. albicans, although filamentous growth is enhanced at higher temperatures, fungal germination has been observed in vivo at 28°C and many studies have implicated a role of filamentation in virulence at this temperature.

“However, in the case of C. albicans, although filamentous growth is enhanced at higher temperatures, fungal germination has been observed in vivo at 28°C and many studies have implicated a role of filamentation in virulence at this temperature (Brennan et al. 2002; Chamilos et al. 2006; Fuchs and Mylonakis 2006; Brothers, Newman and Wheeler 2011).”

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• cites Chamilos et al. 2006Not checked yet

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• cites Fuchs and Mylonakis 2006Not checked yet

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A drawback to using zebrafish is that they are ectothermic and prefer temperatures of 28°C, while humans are endothermic and have a normal body temperature of 36.8 ± 0.4°C.

“A drawback to using zebrafish is that they are ectothermic and prefer temperatures of 28°C, while humans are endothermic and have a normal body temperature of 36.8 ± 0.4°C (Chao et al. 2010; Brothers, Newman and Wheeler 2011; Gratacap and Wheeler 2014).”

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• cites Brothers, Newman and Wheeler 2011Not checked yet

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• cites Gratacap and Wheeler 2014Not checked yet

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Unlike RNAi, the CRISPR-Cas9 system is not limited solely to the silencing or deletion of genes and has been adapted so that it can also be used for editing/modifying the genome via its homology directed repair mechanism, to activate or repress gene expression, to purify regions of genomic DNA and to label genomic DNA for imaging.

“Unlike RNAi, the CRISPR-Cas9 system is not limited solely to the silencing or deletion of genes and has been adapted so that it can also be used for editing/modifying the genome via its homology directed repair mechanism, to activate or repress gene expression, to purify regions of genomic DNA and to label genomic DNA for imaging; see Fig. 3 for a schematic and applications of the CRISPR-Cas9”

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Zalatan et al. adapted the CRISPR-Cas9 system to generate synthetic multigene transcriptional programs by repressing and activating genes to redirect flux through the violacein biosynthetic pathway, which they recreated in S. cerevisiae.

“For example, Zalatan et al. have recently adapted the CRISPR-Cas9 system to generate synthetic multigene transcriptional programs by repressing and activating genes to redirect flux through the violacein biosynthetic pathway (an important metabolic pathway for bacteria) which they recreated in S. cerevisiae.”

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The most commonly used CRISPR Type II system consists of a gRNA and an endonuclease, the CRISPR-associated (Cas) nuclease, Cas9, which cleaves the targeted chromosomal DNA in a site-specific manner triggering endogenous DNA repair systems resulting in genome modification.

“The most commonly used CRISPR Type II system consists of a gRNA and an endonuclease, the CRISPR-associated (Cas) nuclease, Cas9, which cleaves the targeted chromosomal DNA in a site-specific manner triggering endogenous DNA repair systems resulting in genome modification (for an in-depth review, see Doudna and Charpentier 2014; Kim and Kim 2014; Shalem, Sanjana and Zhang 2015).”

• cites Doudna and Charpentier 2014Not checked yet

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• cites Kim and Kim 2014Not checked yet

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• cites Shalem, Sanjana and Zhang 2015 sourceNot checked yet

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Bao et al. (2015) used the CRISPR-Cas9 system to generate multiple gene disruptions in a single step in S. cerevisiae, representing a powerful tool for creating yeast strains with multiple knockouts in less than a week, termed the HI-CRISPR system.

“Bao et al. (2015) on the other hand have used the CRISPR-Cas9 system to generate multiple gene disruptions in a single step in S. cerevisiae representing a powerful tool for creating yeast strains with multiple knockouts in less than a week, and they have termed this the HI-CRISPR system.”

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Tsai et al. (2014) performed comparative genomics on four species of Taphrina fungi and discovered that Taphrina fungi utilized multiple strategies to cope with the host environment that were also found in some yeast species such as aneuploidy of genes.

“For example, Tsai et al. (2014) have recently performed comparative genomics on four species of Taphrina fungi, a plant pathogen that causes plant deformity, and discovered that Taphrina fungi utilized multiple strategies to cope with the host environment that were also found in some yeast species such as aneuploidy of genes.”

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Thus, zebrafish may prove to be a tractable virulence model for real-time visualization of pathogen-host interactions, and although researchers have yet to investigate C. glabrata in a zebrafish model, the need to understand pathogen-host interactions in real time makes zebrafish a serious contender for use in future virulence studies.

“Thus, zebrafish may prove to be a tractable virulence model for real-time visualization of pathogen–host interactions, and although researchers have yet to investigate C. glabrata in a zebrafish model, the need to understand pathogen–host interactions in real time makes zebrafish a serious contender for use in future virulence studies.”

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

With so many fungal genomes readily available, the possibility now exists of performing comparative genomic analysis with C. glabrata to predict evolutionary pathways that may have evolved to aid its survival as a pathogen, and to experimentally validate these predictions.

“With so many fungal genomes readily available, the possibility now exists of performing comparative genomic analysis with C. glabrata to predict evolutionary pathways that may have evolved to aid its survival as a pathogen, and to experimentally validate these predictions.”

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Alternative hosts for virulence studies are emerging and their relative simplicity could be used in global screens facilitating analysis of C. glabrata infections.

“Thus, it can be seen that alternative hosts for virulence studies are emerging and their relative simplicity could be used in global screens facilitating analysis of C. glabrata infections”

May combine two claims.

Based on CAGE analysis, Aoyama et al. predicted 72 C. glabrata genes to contain uORFs, yet none of the 72 S. cerevisiae orthologues have been reported to harbour uORFs; these data strongly suggest that there is regulatory divergence between these two yeasts.

“Instead, based on the CAGE analysis, Aoyama et al. predicted 72 C. glabrata genes to contain uORFs, yet none of the 72 S. cerevisiae orthologues have been reported to harbour uORFs. These data strongly suggest that there is regulatory divergence between these two yeasts (Aoyama et al. 2014).”

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Further advances in next-generation sequencing as well as RNA-seq data in C. glabrata will greatly aid our understanding of the unique changes in transcriptional regulation that occur when C. glabrata encounters a host and the mechanisms underpinning its virulence.

“Further advances in next-generation sequencing as well as RNA-seq data in C. glabrata will greatly aid our understanding of the unique changes in transcriptional regulation that occur when C. glabrata encounters a host and the mechanisms underpinning its virulence.”

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

By 2011 a total of 108 fungal genomes had been annotated, and the growing list of annotated fungal genomes continues to grow as sequencing technologies improve.

“Since the publication of the first genome sequence of a free-living eukaryote, S. cerevisiae, was published 24 years ago by Goffeau et al., by 2011 a total of 108 fungal genomes had been annotated (Goffeau et al. 1996; Haas et al. 2011), and the growing list of annotated fungal genomes continues to grow as sequencing technologies improve.”

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• cites Haas et al. 2011Not checked yet

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May combine two claims.

Candida glabrata NP4 and NP38 were shown to be downregulated upon interaction with human neutrophils, suggesting that the encoded proteins may be important for C. glabrata's role as a pathogen.

“Candida glabrata NP4 and NP38 were also shown to be downregulated upon interaction with human neutrophils, suggesting that the encoded proteins may be important for C. glabrata's role as a pathogen (Linde et al. 2015).”

• cites Linde et al. 2015Not checked yet

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May combine two claims.

Aoyama et al. performed CAGE analysis on RNA isolated from C. glabrata grown in seven different conditions, and this analysis suggested the existence of more than 50 previously unannotated C. glabrata genes as well as confirming the identity of 4316 genes previously listed on the Candida Genome Database.

“Indeed, recently Aoyama et al. performed Cap Analysis Gene Expression (CAGE analysis), a method that was introduced to determine the transcription start sites on a genome-wide scale by isolating and sequencing fragments originating from the 5′ end of RNA transcripts, on RNA isolated from C. glabrata grown in seven different conditions. This analysis suggested the existence of more than 50”

• cites Aoyama et al. 2014 sourceNot checked yet

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May combine two claims.

A host-pathogen interaction network for a C. albicans-zebrafish infection model identified several important proteins related to C. albicans infection that may prove useful drug targets, and identified important immune defensive mechanisms activated in zebrafish in response to C. albicans invasion.

“The power behind this virulence model can be seen by the publication of a host–pathogen interaction network for a C. albicans–zebrafish infection model, which identified several important proteins related to C. albicans infection that may prove useful drug targets. At the same time, the study identified important immune defensive mechanisms activated in zebrafish in response to C. albicans”

• cites Kuo et al. 2013Not checked yet

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• cites Gratacap and Wheeler 2014Not checked yet

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

Three distinct bacterial CRISPR systems have been identified; Type I, II and III, and it is the Type II system that has mainly been adapted for use in artificial gene regulation and genome editing.

“So far three distinct bacterial CRISPR systems have been identified; Type I, II and III, and it is the Type II system that has mainly been adapted for use in artificial gene regulation and genome editing (Kim and Kim 2014; Shalem, Sanjana and Zhang 2015).”

• cites Kim and Kim 2014Not checked yet

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• cites Shalem, Sanjana and Zhang 2015 sourceNot checked yet

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

The CRISPR-Cas9 system has revolutionized and advanced research in the field of artificial gene regulation and genome editing.

“A technology that has revolutionized and advanced research in the field of artificial gene regulation and genome editing is the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system (Doudna and Charpentier 2014).”

• cites Doudna and Charpentier 2014Not checked yet

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May combine two claims.

252 genes in S. cerevisiae have been reported to contain upstream small open reading frames (uORFS) that can inhibit translation of the downstream ORF, and none of the C. glabrata orthologous genes were reported to contain uORFs.

“For example, 252 genes in S. cerevisiae have been reported to contain upstream small open reading frames (uORFS) that can inhibit translation of the downstream ORF by interfering with the initiation of translation from its start codon. None of the C. glabrata orthologous genes were reported to contain uORFs (Aoyama et al. 2014).”

• cites Aoyama et al. 2014 sourceNot checked yet

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May combine two claims.

Both Aoyama et al. (2014) and Linde et al. (2015) found divergence in the transcriptional regulation between C. glabrata and S. cerevisiae, reinforcing the idea that great care should be taken when extrapolating such regulatory data between species.

“Interestingly both Aoyama et al. (2014) and Linde et al. (2015) found divergence in the transcriptional regulation between C. glabrata and S. cerevisiae, thereby reinforcing the idea that great care should be taken when extrapolating such regulatory data between species.”

• cites Aoyama et al. 2014 sourceNot checked yet

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• cites Linde et al. 2015Not checked yet

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May combine two claims.

Linde et al. verified four of the novel genes, C. glabrata NP4, 11, 32 and 38 via RT-PCR, and showed that their expression was upregulated during a pH shift from pH4 to pH8, suggesting that the encoded proteins play a role in helping C. glabrata regulate and/or adapt to changes in environmental pH.

“These workers went on to verify four of the novel genes, C. glabrata NP4, 11, 32 and 38 via RT-PCR, and showed that their expression was upregulated during a pH shift from pH4 to pH8 suggesting that the encoded proteins play a role in helping C. glabrata regulate and/or adapt to changes in environmental pH (Linde et al. 2015).”

• cites Linde et al. 2015Not checked yet

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

CRISPR-Cas systems utilize short guide RNA (gRNA) strands to direct the degradation of foreign DNA that the system has previously encountered by incorporating fragments of foreign DNA into the CRISPR loci to produce the short gRNAs required to degrade homologous sequences.

“Similar to RNAi, CRISPR-Cas systems utilize short guide RNA (gRNA) strands to direct the degradation of foreign DNA that the system has previously encountered by incorporating fragments of foreign DNA into the CRISPR loci to produce the short gRNAs required to degrade homologous sequences (Mali, Esvelt and Church 2013; Gilles and Averof 2014; Laganà, Shasha and Croce 2014).”

• cites Mali, Esvelt and Church 2013Not checked yet

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• cites Gilles and Averof 2014Not checked yet

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• cites Laganà, Shasha and Croce 2014Not checked yet

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May combine two claims.

Potentially, using the HI-CRISPR system a C. glabrata strain with six targeted genes knocked out/disrupted could be created in as little as 12 days, whereas it currently takes several weeks to several months to make a double knockout in C. glabrata.

“Potentially, using the HI-CRISPR system a C. glabrata strain with six targeted genes knocked out/disrupted could be created in as little as 12 days, whereas it currently takes several weeks to several months to make a double knockout in C. glabrata, speeding up research as the construction of knockout/disrupted genes is often a rate-limiting step in advancement.”

Probably not a substantial claim: may not pass S2 (checkable).

Recent advances in the C. glabrata molecular tool box should reveal novel putative drug targets.

“Recent advances in the C. glabrata molecular tool box should aid research into its virulence mechanisms, host–pathogen relationship and reveal novel putative drug targets.”

• AssumptionRests on an assumption: Still today relatively little is known about the molecular basis of virulence of C. glabrata.

Classified as probably not a substantial claim.

Recent advances in the C. glabrata molecular tool box should aid research into its virulence mechanisms.

“Recent advances in the C. glabrata molecular tool box should aid research into its virulence mechanisms, host–pathogen relationship and reveal novel putative drug targets.”

• AssumptionRests on an assumption: Still today relatively little is known about the molecular basis of virulence of C. glabrata.

Classified as probably not a substantial claim.

Making a knockdown construction of an essential gene in C. glabrata will be less time consuming and difficult.

“for instance, making a knockdown construction of an essential gene in C. glabrata will be less time consuming and difficult”

• AssumptionRests on an assumption: Still today relatively little is known about the molecular basis of virulence of C. glabrata.

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

A partial C. glabrata ORFeome is being constructed.

“Additionally, a partial C. glabrata ORFeome is being constructed (Ho and Huvet et al., unpublished, Ho et al., unpublished)”

• cites Ho and Huvet et al., unpublishedNot checked yet

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• cites Ho et al., unpublishedNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

It is likely that a proportion of the essential genes will play an important role in virulence of C. glabrata.

“It is likely that a proportion of the essential genes will play an important role in virulence of C. glabrata.”

• AssumptionRests on an assumption: Still today relatively little is known about the molecular basis of virulence of C. glabrata.

Probably not a substantial claim: may not pass S4 (based).

Once complete, the partial C. glabrata ORFeome will facilitate high-throughput phenotypic screens and basic research.

“and once complete will facilitate high-throughput phenotypic screens and basic research.”