MICROBIAL SPECIES

GOVERNMENT FUNDING

[0001] The present invention was made with government support under Grant Nos. ROA1141893 and R01A1081704, awarded by the National Institutes of Health. The US government has certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63/352,728, filed June 16, 2022, which is incorporated herein by reference.

BACKGROUND

[0003] Candida albicans is a common component of the human microbiome, often found inhabiting mucosal niches such as the gastrointestinal (GI) tract and the skin. However, this species is also a frequent cause of life-threatening systemic infections in the US, and even with treatment with antifungal drugs mortality can reach 40%. Kett, D. H., et. al. Critical care medicine 39, 665-670 (2011). Despite its importance as a human pathogen, genetic analysis of this species has lagged behind those of related yeast species such as Saccharomyces cerevisiae. Reasons for this include the fact that C. albicans isolates are naturally diploid, Butler, G. et al. Nature 459, 657-662, (2009), and while haploid forms have been identified these are not in common use for studies of commensalism or pathogenesis due to their decreased fitness Hickman, M. A. et al. Nature 494, 55-59, (2013). Moreover, C. albicans is a member of the CTG clade (or Seri subclade), where the CTG codon is translated as serine instead of leucine as in the universal genetic code, Krassowski, T. et al. Nature communications 9, 1887, (2018), meaning that constructs often have to be codon-optimized specifically for these species.

[0004] An additional limitation has the paucity of dominant selectable markers available for C. albicans and related CTG clade species. Currently, only one such selectable system is commonly in use in C. albicans, which involves the SAT1/NAT1 gene that provides selection to the antibiotic nourseothricin, Shen, J., et al. Infect Immun 1 , 1239-1242, (2005). Recycling of the SAT1 gene enables multiple C. albicans transformations with this marker, Reuss, O., et al. Gene 341, 119-127 (2004), but this is time consuming and chromosomal rearrangements can occur when site-specific recombination sequences are present in multiple places in the genome, Solis-Escalante, D. et al. FEMS Yeast Res 15, (2015). There is therefore a pressing need to identify additional dominant selectable markers that are complementary to that of SAT1/NAT1.

[0005] One major obstacle to the utilization of alternative selectable markers has been the fact that C. albicans often exhibits high levels of tolerance/resistance to antibiotics used for genetic selection in other yeast species. For example, the aminoglycoside antibiotics hygromycin B and kanamycin/G418 are frequently used for selection in 5. cerevisiae using the selectable markers HphMX and KanMX, respectively, Wach, A., et al. Yeast 10, 1793-1808 (1994), yet C. albicans exhibits high levels of growth on both of these antibiotics. A codon-optimized hygromycin B gene (CaHygB) was previously developed for C. albicans, Basso, L. R., Jr. et al. Yeast 27, 1039-1048, (2010), yet has not been commonly adopted by the field due to high background growth even with high levels of hygromycin B. Overexpression of an IMH3 allele was also shown to provide resistance against mycophenolic acid in C. albicans but has not been adopted by the field due to issues with its implementation, Beckerman, J., et al. Infect Immun 69, 108-114, (2001).

[0006] Multiple studies have examined combinatorial mixtures of antifungal compounds to determine those that exhibit additive or synergistic inhibition of fungal growth. These include those of Vallieres et al. who recently showed that the combination of certain transport inhibitors with aminoglycoside antibiotics had a synergistic effect on inhibiting C. albicans growth, Vallieres, C., et al. Frontiers in microbiology 9, 2355, (2018). In particular, synergistic inhibition occurred when combining hygromycin B with inhibitors of amino acid transport (e.g., quinines) or sulphate transport (e.g., molybdate or chromate), id. These combinations were effective at blocking growth as transport inhibitors led to a decreased pool of amino acids that synergized with the ribosome-targeting aminoglycosides.

SUMMARY OF THE INVENTION

[0007] Candida albicans is a pathobiont fungus that can colonize multiple niches in the human body but is also the frequent cause of both mucosal and systemic disease. A paucity of dominant selectable markers has hindered progress in tool development for genetic manipulation of the organism. One limiting factor for the utilization of dominant selectable markers is that C. albicans is inherently more resistant to antibiotics used for selection in other fungal species. The inventors have shown that the inclusion of suitable adjuvants can enable the use of twoaminoglycoside antibiotics, hygromycin B and G418, for positive selection in C. albicans. Combining these antibiotics with an adjuvant, such as quinine or molybdate, substantially suppress the background growth of C. albicans cellsthereby enabling transformants expressing CaHygB or CaKan markers to be readily identified. It was verified that these adjuvants are not mutagenic in C. albicans and that CaHygB and CaKan markers are orthogonal to the existing marker, NAT1/SAT1 , and so provide complementary tools for enabling the genetic manipulation of C. albicans strains. This work also establishes that adjuvant-based approaches can enable the use of selectable markers that would otherwise be limited by high background growth from susceptible cells.

BRIEF DESCRIPTION OF THE FIGURES

[0008] The present invention may be more readily understood by reference to the following figures, wherein:

[0009] Figures 1A and IB provide graphs showing that quinine (A) and molybdate (B) act in tandem with hygromycin to inhibit C. albicans growth. 96-well checkerboard assays were performed in YPD containing the indicated drug concentrations. Plates were incubated at 30°C with orbital shaking, and ODeoo was recorded for each well every 15 min for 24 h. The relative growth of each well was normalized to the growth of the well with the highest OD.

[0010] Figures 2A-2C provide images showing quinine acts in combination with hygromycin for the selection of a CaHygB marker. (A) Transformation with a linearized construct carrying the CaHygB gene was carried out and plated on YPD containing different combinations of hygromycin and quinine as indicated. Plates were incubated at 30°C for 72 h. (B) The parental SC5314 C. albicans strain and seven colonies from the plate containing 0.6 mg/mL hygromycin + 1.75 mg/mL quinine were subcultured on plates containing different combinations of hygromycin and quinine as indicted. Plates were incubated at 30°C for 24 h. (C) PCR check for successful integration of the CaHygB gene into the C. albicans genome. All seven transformant colonies were positive, confirming the successful integration of CaHygB. The parental SC5314 strain was used as a negative control, and a strain carrying pSFS2A-SATl similarly integrated at the MAL2 promoter was used as a positive control.

[0011] Figure 3 provides graphs showing quinine (A) and molybdate (B) act in tandem with G418 to inhibit C. albicans growth. The 96- well checkerboard assays were performed in YPD containing the indicated drug concentrations. Plates were incubated at 30°C with orbital shaking, and ODeoo was recorded for each well every 15 min for 24 h. The relative growth of each well was normalized to the growth of the well with the highest optical density (OD).

[0012] Figures 4A-4D provide images showing that quinine and molybdate act in combination with G418 for the selection of a CaKan marker. (A) Transformation of a plasmid carrying the CaKan gene was performed and transformants were cultured on YPD medium containing different combinations of G418, quinine, and molybdate as indicated. Plates were incubated at 30°C for 72 h. (B) The parental SC5314 strain and seven transformant colonies (taken from the plate containing 2 mg/mL G418 + 2 mg/mL quinine) were cultured on plates containing G418 and quinine as indicated. Plates were incubated at 30°C for 24 h. (C) In a control SC5314 strain, seven transformants from G418/molybdate selection were subcultured on plates containing different G418 and molybdate concentrations as indicated. Plates were incubated at 30°C for 24 h. (D) PCR analysis of seven transformants from G418/quinine and seven colonies from G418/molybdate were tested and all 14 contained the CaKan cassette correctly integrated at the MAL2 locus. The parental SC5314 strain was used as a negative control and a strain containing a pSFS2A-SATl construct similarly integrated at the MAL2 promoter was used as a positive control.

[0013] Figure 5 provides images showing SAT1, CaHygB, and CaKan are three orthogonal selection markers in C. albicans. C. albicans strains carrying the SAT1 nourseothricin marker, the CaHygB hygromycin marker, the CaKan G418 marker, or no selection marker were grown on YPD, YPD + 0.2 mg/mL NAT, YPD + 0.6 mg/mL hygromycin (+ 1.75 mg/mL quinine), or YPD + 2 mg/mL G418 (+ 2 mg/mL quinine). Only cells carrying the corresponding selection marker grew on the appropriate antibiotic. Plates were incubated at 30°C for 24 h.s.

[0014] Figure 6 provides a graph showing quinine and molybdate are not significantly mutagenic in C. albicans. URA3 heterozygous C. albicans strain was incubated on plates containing different concentrations of adjuvants as indicated. Colonies were resuspended after 72 h of incubation at 30°C and plated to 5-FOA and YPD plates. Mutagenicity was calculated by dividing the number of colonies on 5-FOA plates by the number of colonies on YPD plates multiplied by 10,000. Each condition includes 4 replicates. The significance test was performed with Welch’s t test with a significance value of 0.05.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention provides a method of selecting a recombinant microorganism. The method includes providing a genetically modified microorganism that includes an antibiotic selection marker; contacting the genetically modified microorganism with an effective amount of an antibiotic specific for the antibiotic selection marker and an adjuvant; and selecting the genetically modified microorganism by isolating the microorganisms that survive contact with the antibiotic and adjuvant. Methods of identifying novel antibiotic selection markers are also provided.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which these exemplary embodiments belong. The terminology used in the description herein is for describing particular exemplary embodiments only and is not intended to be limiting of the exemplary embodiments. As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0017] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. [0018] The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.

[0019] The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0020] “Recombinant,” as used herein, refers to an organisms that results from combining genetic material from two different species, such as a host microorganism that has been modified to include DNA from a different organism.

[0021] “Exogenous,” as used herein with reference to a nucleic acid sequence, means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found.

[0022] A "gene, " or a "sequence which encodes " a particular protein, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of one or more appropriate regulatory sequences. A gene of interest can include, but is no way limited to, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the gene sequence. Typically, a polyadenylation signal is provided to terminate transcription of genes inserted into a recombinant virus.

[0023] The term "operably linked," as used herein, refers to the arrangement of various nucleic acid molecule elements relative to each other such that the elements are functionally connected and are able to interact with each other. Such elements may include, without limitation, a promoter, an enhancer, a polyadenylation sequence, one or more introns and/or exons, and a coding sequence of a gene of interest to be expressed. The nucleic acid sequence elements, when operably linked, can act together to modulate the activity of one another, and ultimately may affect the level of expression of the gene of interest, including any of those encoded by the sequences described above.

[0024] An “effective amount,” as used herein, refers to an amount sufficient to achieve an intended result. An “effective amount” includes an amount that is 100% effective in achieving that result, but also includes amounts that are less effective but still exhibit a significant effect. For example, an effective amount of an antibiotic compound used for antibiotic selection is an amount sufficient to kill a sufficient number of microorganisms not having an antibiotic selection marker for microorganisms having that selection marker to be readily separated and identified.

[0025] All scientific and technical terms used in the present application have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present application.

Selecting a Recombinant Microorganism

[0026] In one aspect, the present invention provides a method of selecting a recombinant microorganism. The method includes the steps of providing a genetically modified microorganism that includes an antibiotic selection marker; contacting the genetically modified microorganism with an effective amount of an antibiotic specific for the antibiotic selection marker and an adjuvant; and selecting the genetically modified microorganism by isolating the microorganisms that survive contact with the antibiotic and adjuvant.

[0027] In some embodiments, providing a genetically modified microorganism comprises modifying a microorganism to include an antibiotic selection marker associated with an exogenous gene. Associating the antibiotic selection marker with the exogenous gene results in either both or neither being present in the genetically modified organism. For example, the antibiotic selection marker can be operably linked to the exogenous gene, or it can be expressed on the same plasmid. A vector can be used to genetically modify the microorganism so that it contains one or more antibiotic selectable markers associated with an exogenous gene that permit easy selection of transformed, transfected, transduced, or the like cells that include the exogenous gene. [0028] The expression vector may contain a selectable marker for isolating, identifying or tracking recombinant microorganisms. The expression vector may also provide unique or conveniently located restriction sites to allow severing and/or rearranging portions of the DNA inserts in an expression, and/or promoters or other regulatory elements, which may be operatively linked to the exogenous gene to assure that the exogenous gene is adequately expressed. Accordingly, preparing a recombinant microorganism can include introducing recombinant DNA into microorganisms (e.g., yeast strains) and for obtaining stable transformants. In some embodiments, the exogenous gene is a protein expressing gene.

[0029] The present invention provides a method of selection for genetically modified organisms (e.g., transformants) comprising the steps of: a) introducing a selection marker expression module into a host cell; and b) growing the host cell in the presence of an antibiotic specific for that antibiotic selection marker and an adjuvant, preferably an adjuvant that is synergistic with the antibiotic. Transformed cells including the selection marker can grow in the presence of antibiotic and can thus be separated from microorganisms lacking the antibiotic selection marker, which cannot grow in the presence of the antibiotic and adjuvant.

[0030] In some embodiments, the microorganism is a fungus. "Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0031] Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen el al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920. [0032] In some embodiments, the fungal host cell may be a yeast cell. "Yeast" as used herein includes ascosporog enous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0033] The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces , or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

[0034] The present invention also provides a method for transformation of yeast with a vector that includes an antibiotic selection marker, and where the method comprises transforming yeast with a gene encoding selection marker such that the selection marker integrates into the chromosome of the yeast in a manner which permits detection of the modified recombinant yeast, for example, following restriction digestion of the chromosome. Yeast transformed using this method can include C. albicans and .S', cerevisiae.

[0035] Another method for transferring an antibiotic selection marker associated with an exogenous gene into the yeast by recombinant DNA techniques comprises the steps of: a) operatively linking the selection marker in a plasmid; b) transforming the yeast with the plasmid; c) selecting a transformed yeast of step (b) which expresses selection marker by direct selection on a medium containing an appropriate antibiotic and adjuvant.

[0036] The antibiotic selection marker is a gene that improves the ability of the microorganism to survive in the presence of the antibiotic, and are used to indicate the success of a transfection or other procedure for introducing foreign DNA into a cell. Preferably the antibiotic selection marker is a positive selection marker. In some embodiments, the antibiotic selection marker is one that has been identified for use in a microorganism using the methods described herein. A number of antibiotic selection markers are known to those skilled in the art. For example, the antibiotic selection marker can be an antibiotic resistance gene that produces a protein that provides cells expressing this protein with resistance to one or more antibiotics. Examples of antibiotic selection markers include genes encoding beta-lactamase, the Neo gene from Tn5, the mFabl gene, and URA3.

[0037] Selectable markers (e.g., antibiotic selection markers) for use in fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5 '-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and a Streptomyces hygroscopicus bar gene. In some embodiments, the antibiotic selection marker comprises the CaHygB and/or CaKan antibiotic selection marker.

[0038] In some embodiments, the microorganism comprises the NAT1/SAT1 selection marker. For a description of the NAT1/SAT1 selection marker and its use in selecting recombinant fungi (e.g., C. albicans) using the antibiotic nourseothricin, See U.S. Patent No. 6,562,595, the disclosure of which is incorporated herein by reference.

[0039] In some embodiments, the microorganism comprises a plurality of selection markers for antibiotics. The selection markers are preferably selection markers for different antibiotics. In further embodiments, the selection markers are orthogonal.

[0040] In some embodiments, the adjuvant and the antibiotic have a synergistic inhibitory effect on the microorganism. Those skilled in the art are aware of many different adjuvants that have a synergistic effect with specific antibiotics or antibiotic classes. For example, see Vallieres et al., Frontiers in microbiology 9, 2355, (2018); Moreno-Martinez et al., Scientific reports 5, 16700, (2015); and Bailey et al., mBio., 13(l):e0308421 (2022).

[0041] A variety of different antibiotics can be used to select modified microorganisms including the antibiotic selection marker. The antibiotic should be one that allows the modified microorganisms to escape the cytotoxic effect of the antibiotic based on the selection marker, thereby allowing the modified microorganism to survive and be selected. In some embodiments, the antibiotic is an aminoglycoside antibiotic. For example, in some embodiments, the antibiotic is hygromycin B or a kanamycin antibiotic (e.g., kanamycin A or G418). [0042] A variety of different adjuvants can be used to select the modified microorganism. See Kalan, L. and Wright, G., Expert Rev Mol Med., 13:e5 (2011). The term “adjuvant,” as used herein, refers to compound that improves the effectiveness of one or more of the antibiotics used to select the modified microorganism. In many cases, the adjuvant helps overcome the natural resistance of the microorganism to the antibiotic. Antibiotic adjuvants that rescue antibiotics from resistance are known to those skilled in the art. See Wright, G., Trends Microbiol., 24(1 l):862-871 (2016). For example, the antibiotic can be a (j-lactamase inhibitor, or an outer membrane barrier compromising compound, such as an efflux pump inhibitor or a membrane permeabilizer. In some embodiments, the adjuvant is quinine or molybdate.

[0043] The method includes the step of selecting the genetically modified microorganism by isolating the microorganisms that survive contact with the antibiotic and adjuvant. Methods of selecting recombinant and transformant microorganisms are well known by those skilled in the art. For example, the genetically modified microorganisms can be selected by growing them on culture medium suitable for the microorganism, and isolating surviving colonies of the genetically modified microorganisms.

[0044] In one aspect, the present invention provides a method of selecting a recombinant yeast, comprising: providing a genetically modified yeast including a CaHygB and/or CaKan antibiotic selection marker; contacting the genetically modified yeast with an effective amount of a hygromycin B or kanamycin antibiotic and a quinine or molybdate adjuvant; and selecting the recombinant yeast by isolating the yeast that survive contact with the antibiotic and adjuvant.

[0045] The antibiotic selection marker used should correspond to the antibiotic used for selection of the organism. For example, in some embodiments, the recombinant yeast comprises the antibiotic selection marker CaHygB and the yeast is contacted with hygromycin B, while in other embodiments the recombinant yeast comprises the antibiotic selection marker CaKan and the yeast is contacted with the kanamycin antibiotic G418.

Methods of Identifying an Antibiotic Selection Marker

[0046] A method of identifying an antibiotic selection marker for use in a recombinant microorganism, comprising: modifying a microorganism to include a proposed antibiotic selection marker associated with an exogenous gene; contacting the modified microorganism with an antibiotic specific for the antibiotic selection marker and an adjuvant; and identifying the antibiotic selection marker as effective if the modified microorganism can be easily selected from microorganisms lacking the proposed antibiotic selection marker. This aspect of the method can be used to identify new antibiotic selection markers that can be effectively used in the presence of an adjuvant. Effective use refers to the ability of the antibiotic selection marker to clearly distinguish between modified microorganisms including the marker in comparison with those that do not.

[0047] A proposed antibiotic selection marker may be an antibiotic selection marker which is known, but which has been found to be ineffective with a particular microorganism, or it may be a previously unidentified antibiotic selection marker. When testing a known compound, the test may be useful for determining whether an adjuvant can increase the selection provided by the antibiotic selection marker to a more useful level.

[0048] A significant difference between a method of identifying an antibiotic selection marker and a method of selecting a recombinant microorganism using an antibiotic selection is whether or not it is already known that the antibiotic selection marker is effective before carrying out the method. Accordingly, it is possible to carry out the method of identifying an antibiotic selection marker and obtaining a negative result. Furthermore, it may be desirable to test a plurality of potential antibiotic selection markers using a single assay (e.g., using a multi-well plate) in order to test a large number of candidate compounds simultaneously. For example, in some embodiments, the modified microorganism is contacted with the antibiotic selection marker and an adjuvant in a checkerboard assay.

[0049] The method of identification includes all of the features recited in embodiments of the method of selection previously described herein. For example, in some embodiments, the microorganism is a fungus, while in further embodiments the microorganism is a yeast. In some embodiments, the antibiotic selection marker is one that provides selection when used in combination with an aminoglycoside antibiotic. In further embodiments, the adjuvant and the antibiotic have a synergistic inhibitory effect on the microorganism.

[0050] An example has been included to more clearly describe a particular embodiment of the invention. However, there are a wide variety of other embodiments within the scope of the present invention, which should not be limited to the particular example provided herein. EXAMPLE

An adjuvant-based approach enables the use of dominant HYG and KAN selectable markers in Candida albicans

[0051] The inventors examined whether adjuvants can enable the use of aminoglycoside antibiotics for robust selection of dominant selectable markers in C. albicans. They show that adjuvants can effectively work in tandem with hygromycin B and G418 to reduce the background growth of C. albicans. Moreover, expression of CaHygB or CaKan genes was sufficient for the “clean” selection of C. albicans transformants on a medium containing the corresponding antibiotic supplemented with an adjuvant. CaHygB and CaKan are orthogonal for selection with SAT1/NAT1 , meaning that three dominant selectable systems are now available C. albicans to facilitate genetic analysis of this important human fungal pathogen. This data highlights that the use of appropriate adjuvants with different antibiotic markers is expected to greatly expand the utilization of antibiotics for other microbial species for which high background growth has limited the use of these antibiotics.

Results

Analysis of combinatorial antifungal activities with hygromycin

[0052] The inventors examined whether the inclusion of adjuvants could potentiate the activity of antibiotics to which C. albicans has natural resistance. Previous work showed that hygromycin B inhibits the growth of C. albicans cells, Basso, L. R., Jr. et al. Yeast 27, 1039- 1048, (2010), yet they consistently observed considerable background growth when cells of the standard laboratory strain, SC5314, were grown on YPD at 30°C with high levels of this drug (600 pg/ml). These high levels of background growth limit the use of the CaHygB gene as a robust selectable marker in this species as transformations result in lawns of cells on hygromycin-containing medium (as described below).

[0053] Vallieres et al. recently reported that certain adjuvants can be used in combination with hygromycin B to reduce the growth of susceptible C. albicans cells. Hygromycin B is an aminoglycoside that targets ribosome function and quinine, molybdate and chromate were identified as three compounds that show additive or synergistic interactions with this antibiotic, potentially due to the depletion of the amino acid pool, Vallieres, C., et al. Frontiers in microbiology 9, 2355, (2018). The inventors carried out 96- well checkerboard assays to examine interactions between these chemicals using assays with SC5314 cells grown in microtiter plates in YPD medium at 30°C and absorbance measured every 15 minutes for 24 hours. They found that both quinine and molybdate acted combinatorically with hygromycin to substantially inhibit the growth of C. albicans cells compared to hygromycin or adjuvant alone (Fig. 1A, B).

Adjuvants enable the use of CaHygB as a dominant selectable marker with hygromycin

[0054] The CaHygB gene was codon optimized for C. albicans and encodes for a hygromycin B phosphotransferase activity that inactivates hygromycin, Basso, L. R., Jr. et al. Yeast 27, 1039-1048, (2010). The inventors integrated CaHygB into the pSFS2A vector replacing the SAT1 gene originally present in this vector, Reuss, O., et al. Gene 341, 119-127 (2004). This plasmid can be linearized within the MAL2 promoter that drives the site-specific recombinase used to recycle the Satl marker, which results in integration into the endogenous MAL2 locus (see Methods and Materials). Transformations were carried out using a standard lithium acetate/PEG protocol and transformation mixtures plated to YPD supplemented with hygromycin alone or hygromycin containing different amounts of quinine (0 - 1.75 mg/mL) or molybdate. As shown in Figure 2A, transformation plates containing only hygromycin (0.6 mg/mL) or quinine (up to 1.75 mg/mL) generated lawns of cells where individual colonies were not visible. In contrast, individual colonies were evident on all of the hygromycin plates supplemented with quinine or molybdate, and background growth was essentially abolished on hygromycin plates containing 1.5 or 1.75 mg/mL quinine (Fig. 2A).

[0055] Colonies from the hygromycin + 1.75 mg/mL quinine plate were subcultured on hygromycin plates supplemented with different quinine concentrations, together with the parental SC5314 control. These plates showed that colonies from the transformation plate regrew on each of the test plates while SC5314 control cells grew on hygromycin alone but not on any of the hygromycin + quinine plates (Fig. 2B). PCRs were performed on the transformation colonies and confirmed that all of the test colonies from hygromycin+quinine plates contained the construct correctly integrated at the MAL2 locus (Fig. 2C).

[0056] These results demonstrate that medium containing hygromycin alone is limited in its use for direct selection of C. albicans transformants expressing the CaHygB marker. However, the combination of quinine or molybdate with hygromycin enables the robust selection of transformant colonies with little background growth.

Analysis of combinatorial antifungal activities with G418

[0057] Given that adjuvants can decrease the growth of susceptible C. albicans cells on hygromycin-containing medium, the inventors examined whether adjuvants could similarly enable selection with a second aminoglycoside antibiotic, G418. Many fungi are susceptible to G418 (geneticin) which is closely related to kanamycin A, and the KanMX module has been extensively used as a selectable marker for this antibiotic in .S’, cerevisiae, Wach, A., et al. Yeast 10, 1793-1808 (1994). The KanMX module utilizes the kan ^R gene from E. coli transposon Tn903 to provide resistance against G418, id.

[0058] To determine whether compounds that synergize with hygromycin also work in tandem with G418, checkerboard assays were performed with SC5314 cells grown in the presence of G418 together with varying concentrations of quinine or molybdate. Strikingly, both of these compounds worked as adjuvants in combination with G418 (Fig. 3A,B), demonstrating that quinine is effective when used with two distinct aminoglycosides.

Adjuvants enable the use of CaKan as a dominant selectable marker for G418

[0059] There are currently no reports of G418 having been used for antibiotic selection in C. albicans. To determine if the KanR gene from the Tn903 transposon is functional in C. albicans, the inventors codon optimized this gene for this species and used the resulting CaKan to replace the Satl marker in pSFS2A. This construct was again targeted for integration at the MAL2 locus by linearization within the pMAL2 promoter and transformants grown on YPD plates supplemented with G418 either with or without the addition of quinine or molybdate. As shown in Figure 4A the inclusion of either quinine or molybdate was found to result in a substantial reduction in background growth on G418 transformation plates compared to plating on G418 alone. Thus, G418+2 mg/mL quinine or G418+0.5-1 mg/mL molybdate gave very low background growth with which potential transformation colonies were clearly evident (Fig. 4A). Note that colonies grown on molybdate containing media were consistently a darker color, but form the typical off-white colonies when re-struck on media lacking molybdate. [0060] Colonies from G418+quinine or G418+molybdate transformation plates were subcultured onto G418-containing plates with or without these adjuvants, or onto adjuvant- only containing plates. In each case, colonies from transformations grew on G418+adjuvant plates whereas SC5314 control cells grew on G418- or adjuvant-containing plates only, but not on plates containing both antibiotic plus adjuvant (Fig. 4B). To confirm that colonies from G418+adjuvant transformations contained the CaS2m-pSFS2A construct, 7 random colonies were analyzed from both G418+quinine and G418+molybdate selection conditions. PCR analysis of these colonies confirmed that each carried the CaKan construct correctly integrated at the MAL2 locus, demonstrating that this selection method can be successfully used for genetic studies in C. albicans.

CaHygB and CaKan are orthologous markers to SAT1/NAT1 in C. albicans

[0061] To establish that CaHygB and CaKan can be used as complementary markers to SAT1/NAT1 in C. albicans, the inventors analyzed the growth of SC5314 cells carrying each of these markers (as well as control SC5314 cells) on YPD, YPD+nourseothricin, YPD+hygromycin (-(-adjuvant), and YPD+G418(+adjuvant). As shown in Figure 5, only cells carrying a dominant selectable marker were able to grow on the corresponding antibioticcontaining medium. Thus, CaHygB, CaKan and SAT1/NAT1 represent three orthologous markers that can now be robustly used for genetic selection experiments in C. albicans, including the robust identification of transformants.

Media containing adjuvants are not mutagenic to C. albicans

[0062] To examine if the antibiotic selection conditions are mutagenic, the inventors used a counter-selection assay using a C. albicans strain heterozygous for URA3. Loss of URA3 enables growth on medium containing 5-FOA and can therefore be used to monitor levels of mutagenesis. SC5314 cells were grown on plates containing various concentrations of adjuvants at 30°C for 72 hours and replated on SCD plate supplemented with 5FOA and YPD plates. There was no significant difference in the proportion of cells that were 5-FOA-resistant between control and adjuvant containing conditions (Fig. 6). These results establish that quinine and molybdate are not highly mutagenic towards C. albicans and can therefore be adopted for genetic selection assays.

Discussion [0063] The study of C. albicans has lagged considerably behind those of model yeast such as .S', cerevisiae, in large part due to a more limited set of genetic tools. While new experimental approaches continue to expand the toolset for C. albicans, including several CRISPR-based methods for more facile engineering of the species, Uthayakumar, D., et al. Front Genome Ed 2, 606281, (2020), there remain a number of technical limitations compared to model fungi. This includes the fact that only a single dominant selection system, SAT1/NAT1 for growth on nourseothricin, has been widely adopted by the field, whereas multiple such markers typically exist for other fungal species with 17 such markers reported for .S’, cerevisiae, Solis-Escalante, D. et al. FEMS Yeast Res 13, 126-139, (2013). The presence of only one dominant marker for C. albicans is particularly limiting given that isolates are diploid so that this marker often has to be recycled for gene deletions in clinical isolates where auxotrophic derivatives are not readily available.

[0064] Here, we report that CaHygB or CaKan markers can be robustly used as genetic markers in C. albicans when cells are grown on the appropriate antibiotic (hygromycin B or G418, respectively) and when suitable adjuvants are present by reducing the level of background growth on the plate. CaHygB was reported as a dominant marker for C. albicans in 2010, Basso, L. R., Jr. et al. Yeast 27, 1039-1048, (2010), yet has not been widely adopted by the field which presumably reflects the high levels of background growth observed even with high levels of hygromycin (see Figure 2). A gene providing resistance to G418/kanamycin has not been previously reported as a selectable marker for C. albicans yet the KanMX gene has been extensively used with this antibiotic in .S', cerevisiae, Siewers, V. Methods Mol Biol 1152, 3- 15, (2014). We now demonstrate that CaKan, which is a codon-optimized version of the bacterial Tn 903 phosphotransferase ¹¹ , can be utilized in C. albicans by culturing cells on G418 together with appropriate adjuvants. Transformants carrying CaHygB or CaKan can therefore be cleanly selected using this combinatorial approach, and both CaHygB and CaKan are orthologous to SAT1/NAT1 so that three independent antibiotic markers are now available for this species.

[0065] Both hygromycin and G418 are aminoglycosides that inhibit translation by targeting the ribosome, and previous studies indicated that hygromycin activity against C. albicans is synergistic when combined with certain transport inhibitors, Vallieres, C., et al. Frontiers in microbiology 9, 2355, (2018). The two adjuvants utilized in the current work were molybdate, a sulfate transport inhibitor, Moreno-Martinez, E., et al. Scientific reports 5, 16700, (2015), and quinine, an amino acid transport inhibitor, Khozoie, C., et al. J Biol Chem 284, 17968- 17974, (2009), and both showed combinatorial inhibition of C. albicans growth in our assays. Certain transport inhibitors can synergize with aminoglycosides due to an increased rate of translational errors, Moreno-Martinez, E., et al. Scientific reports 5, 16700, (2015). Several other transport inhibitors such as chromate have also been shown to synergize with aminoglycosides suggesting they could be used as alternatives to quinine/molybdate for enabling antibiotic selection, id. However, we note that both quinine and molybdate are inexpensive and did not cause an increased rate of mutagenicity in C. albicans, making them suitable for genetic selection assays.

[0066] In addition to the development of key reagents for C. albicans research, our study reveals that suitable adjuvant treatment will enable the use of antibiotic markers for other microbial species. We highlight that the major limitation with using aminoglycosides such as hygromycin and G418 in C. albicans was not the lack of a functional antibiotic resistance gene for selection, but the high levels of background growth when using large numbers of cells with these antibiotics. Thus, adjuvants that promote antibiotic efficacy will ensure that these antibiotics are suitable for development with selectable markers. Future studies will look to determine if other dominant selectable markers can now be established for use in C. albicans or other CTG clade species and to more broadly establish combinatorial agents for (robust) selection of genetically marked strains.

Materials and Methods

[0067] Strains and media: C. albicans strain SC5314 was used in this study. All strains were grown at 30°C in YPD medium (10 g/L yeast extract (Gibco), 10 g/L peptone (Gibco) and 20 g/L glucose (Fisher Scientific)). Hygromycin B (GoldBio) and G418 (GoldBio) were dissolved in water and added at the concentrations indicated in the text. Nourseothricin (Jena BioScience) was used at 200 pg/mL.

Plasmids:

[0068] pSFS2A-CaHygB: The CaHygB gene along with the TEF2 promoter and ACT1 terminator was amplified from pYM70 using oligos 1653 and 1651. A 716bp fragment of SFS2A extending from just upstream of the Hindlll site in the FLP recombinase gene through its ACT1 terminator was amplified using oligos 1669 and 1652. These two fragments were fused by PCR using overlapping sequence introduced by oligos 1652 and 1653 by mixing and amplifying with oligos 1669 and 1651. This fusion product was then digested with Hindlll and PstI and cloned into pSFS2A digested with same to generate pRB195. To remove an Xhol restriction site in the TEF2 promoter of pRB195 the plasmid was digested with Xhol, the single stranded overhangs filled with the Klenow fragment of T7 DNA polymerase and ligated to create pRB196. To remove a SacI site in the CaHygB gene, a silent T285G substitution was made using the mutagenic oligos, to generate pRB197.

[0069] pSFS2A-CaKan: Oligos 7128 and 7129 were used to amplify all of pSFS2A aside from the Satl ORF and incorporate Bsal restriction sites on each end. The Candida optomized KanR gene (Twist BioScience - pRB1799) was amplified using oligos 7126/7127 which incorporated Bsal sites to generate complementary overlaps to those on the plasmid amplicon. These two fragments were digested and ligated using the NEB Golden Gate Assembly protocol.

[0070] C. albicans transformations: Yeast transformations were performed using a lithium acetate/PEG method as described, Hemday, A. D., et al. Methods Enzymol 470, 737-758, (2010). pSFS2A, pSFS2A-CaHygB and pSFS2A-CaKan plasmids were linearized with BsrGI to target constructs for integration at the C. albicans MAL2 promoter.

[0071] Checkerboard assay: Checkerboard assays were performed as described by EUCAST guidelines, Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST), Clin Microbiol Infect 14, 398-405, (2008). 100 pL of a drug containing combination, in water, was mixed with C. albicans cells in 100 pL of 2xYPD medium (0.02 ODeoo = 10 ⁵ cells) and transferred to each well in a 96-well microtiter plate. Plates were incubated in BioTek Epoch2 microplate spectrophotometer at 30 °C for 24 hours with orbital shaking and absorbance measured at ODeoo-

[0072] Mutagenicity assay: A C. albicans SC5314 strain heterozygous for UR A3 (RBY117728) was utilized for mutagenicity assays. 100 pL of cell suspension (5xl0 ^-5 ODeoo) were first grown on YPD plates supplemented with various concentrations of adjuvants at 30°C for 72 hours. On 2 mg/mL Quinine supplemented YPD plates, 100 pL of 5x10 ⁴ (or 5x10 ³ ) ODeoo cell suspension was plated and incubated at 30°C for 72 hours due to delayed growth. Colonies were picked into cell suspension of 0.5 ODeoo, and 100 pL of cell suspension was plated on SCD plates supplemented with 0.8mg/mL of 5FOA. Same cell suspension was diluted to 5xl0 ^-5 ODeoo, and 100 pL of solution was plated on YPD plates. Colonies were counted after 3 days of incubation at 30°C.

[0073] The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood there from. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.