Claims:We claim:

1. A genetically engineered Candida albicans that is live attenuated, comprising a double deletion of target genes responsible for DNA repair.

2. The genetically engineered Candida albicans as claimed in claim 1, wherein the target genes are selected from WSS1 and RAD51.

3. A vector for producing genetically engineered Candida albicans as claimed in claim 1, comprising:
(i) SAT1 flipper cassette, said cassette comprising SAT1 nourseothricin resistance gene under the control of a Maltose promoter and a recombinase FLP gene; and
(ii) upstream and downstream sequences of RAD51 and WSS1 genes flanking the SAT1 flipper cassette.

4. A method of producing a genetically engineered Candida albicans, comprising the step of:
(i) preparing a deletion construct vector pNA1684 comprising a downstream fragment of Sequence ID No.1, an upstream fragment of Sequence ID No.2 of the target WSS1 gene flanking a SAT-1 flipper cassette;
(ii) preparing a deletion construct vector pNA1686 comprising a downstream fragment of Sequence ID No.1, an upstream fragment of Sequence ID No.3 of the target WSS1 gene flanking a SAT-1flipper cassette;
(iii) preparing a deletion construct vector pNA1722 comprising a downstream fragment of Sequence ID No.4 and an upstream fragment of Sequence ID No.5 of the target RAD51 gene flanking a SAT-1 flipper cassette;
(iv) preparing a deletion construct vector pNA1723 comprising a downstream fragment of Sequence ID No.4, an upstream fragment of Sequence ID No.6 of the target RAD51 gene flanking a SAT-1 flipper cassette;
(v) digestion of the restriction site of the deletion construct vectors by KpnI-SacI restriction enzymes at 37 ?C for 6 hrs, followed by transformation of the digested linear DNA fragments into wild type Sc5314 C. albicans strain ; and
(vi) homologous deletion of the target genes.

5. A set of forward primers represented by Sequence ID No. 7, Sequence ID No.9, Sequence ID No.12, Sequence ID No.14 and a set of reverse primers of Sequence ID No. 8, Sequence ID No.10, Sequence ID No.11, Sequence ID No.13, Sequence ID No.15, Sequence ID No.16 for producing genetically engineered Candida albicans by the method as claimed in claim 4.

6. The method as claimed in claim 4, wherein the preparation of pNA1684 deletion construct vector comprises:
(a) amplifying downstream fragment represented by Sequence ID No. 1 of the target gene WSS1 from the Candida albicans genomic DNA by a forward primer of Sequence ID No.7 and reverse primer of Sequence ID No.8; and cloning the amplified downstream fragment into the SacII-SacI site of a pSFS2 vector; and
(b) amplifying the upstream fragment of Sequence ID No. 2 of the target gene WSS1 from the Candida albicans genomic DNA by a forward primer of Sequence ID No.9 and reverse primers of Sequence ID No. 10 and cloning the upstream fragment of the target gene WSS1 into KpnI-XhoI site of the vector in step (a) comprising the downstream sequence of Sequence ID No.1 of WSS1 gene in step (a).

7. The method as claimed in claim 4, wherein the preparation of pNA1686 deletion construct vector comprises:
(a) amplifying downstream fragment represented by Sequence ID No. 1 of the target gene WSS1 from the Candida albicans genomic DNA by a forward primer of Sequence ID No.7 and reverse primer of Sequence ID No.8; and cloning the amplified downstream fragment into the SacII-SacI site of a pSFS2 vector; and
(b) amplifying the upstream fragment of Sequence ID No. 3 of the target gene WSS1 from the Candida albicans genomic DNA by a forward primer of Sequence ID No.9 and reverse primers of Sequence ID No. 11 and cloning the upstream fragment of the target gene WSS1 into the KpnI-XhoI sites of the vector in step (a) comprising the downstream sequence of Sequence ID No.1 of WSS1 gene in step (a).

8. The method as claimed in claim 4, wherein the preparation of pNA1722 deletion construct vector comprises:
(a) amplifying downstream fragment of Sequence ID No. 4 of the target gene RAD51 from the Candida albicans genomic DNA by a forward primer of Sequence ID No.12 and reverse primer of Sequence ID No.13; and cloning the amplified downstream fragment into the SacII-SacI site of pSFS2 vector; and
(b) amplifying the upstream fragment of Sequence ID No.5 of the target gene RAD51 from the Candida albicans genomic DNA by a forward primer of Sequence ID No.14 and reverse primer of Sequence ID No 15 and cloning the upstream fragment of the target gene RAD51 in the KpnI-XhoI site of the vector in step (a) comprising the downstream sequence of Sequence ID No. 4 of RAD51 gene in step (a) generating a deletion construct of RAD51.

9. The method as claimed in claim 4, wherein the preparation of pNA1723 deletion construct vector comprises:
(a) amplifying downstream fragment of Sequence ID No. 4 of the target gene RAD51 from the Candida albicans genomic DNA by a forward primer of Sequence ID No.12 and reverse primer of Sequence ID No.13; and cloning the amplified downstream fragment into the SacII-SacI site of pSFS2 vector;
(b) amplifying the upstream fragment of Sequence ID No.6 of the target gene RAD51 from the Candida albicans genomic DNA by a forward primer of Sequence ID No.14 and reverse primer of Sequence ID No. 16 and cloning the upstream fragment of the target gene RAD51 in the KpnI-XhoI site of the vector in step (a) comprising the downstream sequence of Sequence ID No. 4 of RAD51 gene in step (a) generating a deletion construct of RAD51.

10. A set of forward primers represented by Sequence ID No.7, Sequence ID No.9, Sequence ID No.12, Sequence ID No.14 and a set of reverse primers of Sequence ID No. 8, Sequence ID No.10, Sequence ID No.11, Sequence ID No.13, Sequence ID No.15, Sequence ID No.16 for producing genetically engineered Candida albicans as claimed in claim 1.
, Description:FIELD OF THE INVENTION:
[001] The present invention relates to a generation of a live attenuated knockout strain of Candida albicans (CNA94) and demonstration of its avirulency in in vivo model.

BACKGROUND OF THE INVENTION:
[002] In addition to viruses and bacteria, fungi are the other microorganisms, that are either equally or more dangerous to human health. Out of about 4 million diverse fungal species, ~300 species have been identified as human pathogens to cause diseases (1). Candidiasis, aspergillosis, mucormycosis, and cryptococcosis are standout fungal infections. Despite the currently available diagnosis and treatment, about 1.5 million deaths occur per year accounting for fungal infections worldwide, and the fatality rate is very similar to be caused by tuberculosis or HIV and is more than malaria or breast or prostate cancer (2). Noteworthy to point out that Candida species alone are primarily accountable for the bulk of these fungal infections. The superficial mucosal candidiasis (vulvovaginal candidiasis and oropharyngeal candidiasis) to life-threatening systemic infections (candidemia or fungemia) are inflicted by Candida species. Systemic candidiasis is frequently reported as a consequence of intestinal dysbiosis, impaired host immunity, and high-risk associated medical therapy like immunosuppressive therapy, central venous catheters, or surgical intervention (3-6). Candidiasis represents the 4th and 6th most common healthcare-associated bloodstream infections in the United States and Europe, respectively (7,8). Candidemia is estimated to afflict ~400,000 patients per year worldwide (9). The late and poor diagnosis of invasive candidiasis has only contributed to the rise in multi-organ failures by septicemia-associated fatality. C. albicans is by far the major cause of infections among all Candida species, trailed by C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei. Moreover, the recent emergence of drug resistant C. auris has challenged the existing healthcare system (11). Candida albicans is a pathobiont which lives on human organs as a commensal, but they often cause superficial to blood stream infections in immunosuppressed individuals. The occurrence of candidiasis, the fungal infections caused by Candida species, has considerably increased in the last few decades. Alarming bloodstream fungal infections are associated with a higher rate of morbidity and mortality, and represent major healthcare problems with high socio-economic impact worldwide. Much of the available information and strategy for overcoming bacterial and viral infection has also been deployed for curbing the fungal invasion. As of today, chemotherapy is the sole available option for overcoming fungal diseases. However, overcoming and eliminating the fungal infection has remained a domain of concern for healthcare professionals, globally. Vaccines play a critical role in preventing deaths, hospitalization, and the spreading of diseases caused by infectious agents. As a protective and preventive strategy, various vaccination programs have been placed in several countries and achieved remarkable success in reducing morbidity and mortality associated with various infections. Based on the studies of host-fungal interaction, in recent years, several groups have reported the immunogenicity and efficacy of different kinds of potential vaccine candidates against Candida in animal models (12-14). Even few potential vaccines have been reported to be effective and safe in initial clinical trials (15, 16). For example, NDV-3A (alum as an adjuvant and the N-terminus of a recombinant Als3 protein of C. albicans as antigen) is the first vaccine to demonstrate preclinical efficacy in protecting from diseases caused by both fungal and bacterial pathogens (novadigm.net). According to a phase I clinical trial, the NDV vaccine was safe and effectively induced antibody and T-cell immune responses in healthy individuals (15). The phase 2 randomized, double-blind, placebo-controlled clinical trial has also been recently completed, and the study found that a single dose of NDV-3A was also safe and effective in patients with recurrent vulvovaginal candidiasis (RVVC), and the vaccine reduced the frequency of symptomatic episodes of vulvovaginal candidiasis for up to 12 months in women aged <40 years (17). Another vaccine being conducted in the clinical trial (Phase I) is on the Sap2 protein of C. albicans in virosomal formulation against RVVC (PEV7, Pevion Biotech AG, Switzerland) (16,18). It appears that PEV7 was safe, effective, and specific and functional B cell memory in 100 % of vaccinated individuals were detected (www.pevion.com). The clinical studies taking larger cohorts including immunosuppressed individuals or at least to individuals receiving corticosteroids and antibiotics are yet to be carried out. Secondly, these potential vaccine candidates seem to protect superficial infection and not blood stream infections, those are more fatal. To sum of it, till today there is no approved vaccine against any fungal infections for clinical use.

[003] Live attenuated vaccine strategy is being used against bacteria and viruses successfully. Live attenuated vaccine is nothing but the whole of pathogen that has just been "weakened" (attenuated) such that it elicits enough protective immune response but does not cause disease in healthy people. It causes a very mild infection that ensures a long-lasting protective immune memory. The concept of live attenuated vaccines came from Edward Jenner's best known Vaccinia virus, a live virus that causes cowpox in cattle but provides cross-protection to smallpox in humans (19). Live-attenuated vaccine strategy is commonly and effectively used in combating viral diseases such as influenza, polio, mumps, rubella, measles, varicella, and rotavirus (20,21). Such approaches have been taken to develop a vaccine against SARS-COV2 also, and it appears to be the lifesaver against COVID19 (22). On the same line, several studies have reported the ability of various attenuated strains of C. albicans to protect against candidiasis. PCA-2 is an echinocandin resistant C. albicans mutant derived from the original 3153A strain. Mice immunized with PCA-2 elicit an innate immune response by increasing the number of peripheral blood polymorphonuclear cells (PMNs) with high candidacidal activity. Consequently, an adoptive transfer of macrophage cells from PCA-2 administered mice conferred substantial protection against subsequent infection (23). Another study evaluated the vaccine potential of CM1613 (deleted in the Mitogen Activated Protein Kinase MKC1), CNC13 (deleted in the MAP-kinase HOG1), and the morphological mutant 92’ attenuated strains in a murine model of invasive candidiasis (24). However, the study suggested that only the CNC13 mutant was able to protect ~60–70% of mice to a subsequent lethal dose wild-type challenge by eliciting both cellular and humoral responses. Ecm33 is a GPI protein involved in the synthesis and preservation of the fungus cell wall. An ecm33? mutant (RML2U) that noted with altered cell wall composition was defective in its interaction with endothelial and epithelial cells, and was shown to protect vaccinated BALB/c mice (25). Similarly, the double-deletion mutant cph1?/efg1? C. albicans is avirulent as it is locked in the yeast form. Although the cph1?/efg1? mutant cells proliferate in infected mice rather than being cleared by the host immune system, its immunization only partially protects mice from systemic infections upon the lethal dose of virulent challenge. Even its booster dose did not improve the degree of protection (26). Despite these advances, most of the avirulent strains are less efficient in protecting fungal infections and have failed to reach a clinical trial stage. Therefore, a potent live attenuated vaccine strain is yet to be identified that will be safe and highly protective against Candida infections.

OBJECTS OF THE INVENTION:

[004] It is therefore an object of the present disclosure to propose a genetically engineered Candida albicans as a potential whole cell vaccine candidate for treating fungal blood stream infection.

[005] Another object of the present disclosure is to generate a knockout strain with homozygous deletions of RAD51 and WSS1 genes in C. albicans.

[006] Yet another object of the present disclosure is to characterize its morphological transition, growth and genome stability.

[007] Still another object of the present disclosure is to decipher the pathogenic potential of the knockout strain in the development of systemic candidiasis in in vivo model.

[008] These and other objects and advantages of the invention will be apparent from the ensuing description and the accompanying drawings.

SUMMARY OF THE INVENTION:

[009] Accordingly, the present invention provides a genetically engineered Candida albicans, comprising a double deletion of target genes involved in DNA repair.
[0010] In one of the feature of the present invention, the target genes are WSS1 and RAD51.

[0011] In another feature of the present invention, vectors for producing genetically engineered Candida albicans, having SAT1 flipper cassette comprising SAT1 encoding nourseothricin resistance gene under the control of a Maltose promoter and a recombinase FLP gene is provided. The vector comprises the upstream and downstream sequences of RAD51 and WSS1 genes flanking the SAT1 flipper cassette.

[0012] In yet another feature of the present invention, set of forward primers and a set of reverse primers of the mentioned genes for producing genetically engineered Candida albicans are provided.

[0013] In still another feature of the present invention, a method of producing a genetically engineered Candida albicans by targeting the mentioned genes is provided.

[0014] In yet another feature to demonstrate avirlency of the knockout strain and compare its pathogenicity with the wild type C. albicans in an in vivo model.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:

[0015] It is to be noted, however, that the appended drawings illustrate only typical embodiments of the present subject matter and are therefore not to be considered for limiting of its scope, for the invention may admit to other equally effective embodiments. The detailed description is described with reference to the accompanying figures. Some embodiments of system or methods in accordance with embodiments of the present subject matter are now described, by way of example, and with reference to the accompanying figures, in which:

Figure 1 (a) illustrates SAT1 fliper cassettes in deletion vector constructs (A) pNA1684 and pNA1686 generated for deletion of WSS1 gene and (B) pNA1722 and pNA1723 generated for deletion of RAD51 gene of C.albicans. Figure 1(b) depicts two vector constructs for each gene WSS1 (A) and RAD51 (B) genes of C. albicans.

Figure 2 is a representative flow chart depicting generation of CNA94 C. albicans strain.

Figure 3 illustrates comparison between the phenotype of the wild strain C. albicans and genetically modified knock out strain of C. albicans (CNA94). (A) Growth rate measurement of strains of C. albicans in liquid culture media, (B) Temperature sensitivity of genetically modified C. albicans (CNA94), (C) Chromosomal instability assay by pulse field gel electrophoresis.

Figure 4 depicts morphological transition of wild type strain and genetically modified strain of C. albicans. (A) Observation of strains under microscope with and without genotoxic stress (B) Filamentation induced by spider containing media (C) Filamentation induced by serum containing media.

Figure 5 shows avirulency of genetically modified strain of C. albicans (CNA94).

DETAILED DESCRIPTION OF THE INVENTION:

[0016] The term “knock–out strain” implies a strain in which one or more genes of the organism is made inoperative or deleted. In the context of the present disclosure the genes are knocked out or deleted from the genome of the organism.

[0017] The term “upstream fragment” implies 5’ end of a DNA strand for the gene and “downstream fragment implies the 3’end of the strand.

[0018] The term “live attenuated” implies a disease-causing microorganism that is weakened in a laboratory so it cannot cause disease. Live attenuated microorganisms are often used as vaccines because, although weakened, they can stimulate a strong immune response.”

[0019] According, to the present disclosure there is provided a genetically engineered Candida albicans, comprising a double deletion of target genes responsible for DNA repair.

[0020] In an embodiment of the present disclosure, the target genes are RAD51 and WSS1.

[0021] Genomic instability leads to various cellular defects and consequently to cell death and diseases. Cells intrinsically possess sophisticated multifactorial DNA damage response pathways that collectively function to suppress the deleterious consequences of DNA damages and maintain genome stability. DNA-protein crosslinks (DPCs) repair is one such pathways (27, 28). DPCs are ubiquitous DNA lesions where a protein moiety is covalently linked to DNA thereby block the progression of replication and transcription, therefore they are highly cytotoxic. DPCs are produced by agents like IR, UV, aldehydes, heavy metal ions, anticancer drugs, and inhibitors of DNA-metabolizing enzymes such as DNA methyltransferase (DNMT) and topoisomerase (Top). Studies from S.cerevisiae and humans suggest that canonical DNA repair pathways like homologous recombination (HR) and nucleotide excision repair (NER) are required for cell survival in the presence of DPCs. While both of these pathways target specifically the DNA component of the DPC, recent reports suggest existence of a protease dependent pathway that specifically removes the protein part of DPCs. Eukaryotic cells carry at least one dedicated DPC protease that diminishes the bulkiness of DPCs. Metalloprotease Wss1 in S. cerevisiae and its mammalian homologue SPRTN function as the DPC specific protease. In S. cerevisiae, the RAD51-dependent pathway of recombination is the most efficient pathway for gene conversion, and is also required for repair of most double strand break repair in mitotic cells.

[0022] The present disclosure delineate roles of these pathways in DPCR and pathogenicity of C. albicans, several knockout strains including CNA94 were generated which contains disruption in RAD51 and WSS1 genes.

[0023] By using homologous recombination strategy and a SAT1 flipper cassette (4,29), a nourseothricin marker gene (SAT1) was inserted into the respective genes in the chromosomes. This facilitated loss function of RAD51 and WSS1 genes. After obtaining the knockout strain, its morphological, growth, ability to maintain genome stability, other phenotypes and pathogenicity were compared those with the parental C. albicans strain (Sc5314).

[0024] The present disclosure also provides a method of producing a genetically engineered Candida albicans strain CNA94 via homologous recombination strategy and a SAT1 flipper cassette.
[0025] The method of producing a genetically engineered Candida albicans strain CNA94 comprises the steps of :
(i) preparing a deletion construct vector pNA1684 comprising a downstream fragment of Sequence ID No.1 and an upstream fragment of Sequence ID No.2 of the target WSS1 gene flanking a SAT1 flipper cassette;
(ii) preparing a deletion construct vector pNA1686 comprising a downstream fragment of Sequence ID No.1 and an upstream fragment of Sequence ID No.3 of the target WSS1 gene flanking a SAT1 flipper cassette;
(iii) preparing a deletion construct vector pNA1722 comprising a downstream fragment of Sequence ID No.4 and an upstream fragment of Sequence ID No.5 of the target RAD51 gene flanking a SAT1 flipper cassette;
(iv) preparing a deletion construct vector pNA1723 comprising a downstream fragment of Sequence ID No.4 and an upstream fragment of Sequence ID No.6 of the target RAD51 gene flanking a SAT1 flipper cassette;
(v) digestion of the restriction site of the deletion construct vectors, followed by transformation of the digested linear DNA fragments to wild type Sc5314 C. albicans strain by a modified lithium acetate method.
(vi) deletion of the target genes by SAT1 flipper cassette occurs by homologous recombination of gene specific sequence present in the deletion constructs and the chromosomal locus.

[0026] In another embodiment of the present disclosure, vectors comprising SAT1 nourseothricin resistance gene (SAT1) under the control of a Maltose promoter and a recombinase FLP gene whose expression is driven from the Maltose promoter gene are provided. The flanking sites of the vectors comprise the amplified upstream and downstream sequences of RAD51 and WSS1.

[0027] In yet another embodiment of the present disclosure, primers for amplifying and confirmation of cloned upstream and downstream sequences of specific genes from Sc5314 genomic DNA are provided.

EXAMPLES:

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

[0029] Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may apply.

Materials and methods:
[0030] The source and geographical origin of the biological materials used in the present invention are given below:
[0031] The wild type strain Candida albicans Sc5314 used in the present disclosure is: Candida albicans (Robin) Berkhout (ATCC MYA-2876).

[0032] The vectors constructs for target genes were synthesized in the laboratory. Primer sequences were designed based on the genome sequence of Sc5314 strain in the Candida genome database.
[0033] The plasmid pSFS2 was obtained from the Institute for Molecular Infection Biology, Germany.

[0034] Restriction enzymes, customised primers, nourseothricin antibiotics were purchased from NEB, IDT and Biotech Desk, respectively.

Example I

[0035] PCR Amplification of the upstream and downstream fragments of Genomic DNA isolated from Candida albicans SC5314 and construction of plasmids, pNA1684, pNA1686, pNA1722 and pNA1723 constructs

[0036] C. albicans possess diploid genome. To facilitate efficient homologous deletion in C. albicans, a modified protocol as described before by the inventors were employed (4, 28).

[0037] Two deletion constructs for each RAD51 and WSS1 genes were generated with different length of upstream sequences but having common downstream sequence (Figure 1).
[0038] Primer sequences were designed based on the genome sequence of Sc5314 strain in the Candida genome database.

[0039] First, the downstream fragment of Sequence ID No. 1 of the WSS1 gene was amplified from Sc5314 genomic DNA by using primers NAP544 of Sequence ID No.7 and NAP545 of Sequence ID No.8 and cloned into the SacII-SacI site of vector pSFS2. Two upstream sequences of Sequence ID No. 2 and Sequence ID No.3 were amplified by using forward primer NAP541 of Sequence ID no. 9 with reverse primers NAP543 of Sequence ID No.10 and NAP542 of Sequence ID No.11, respectively, and then cloned into the KpnI-XhoI sites of the same vector which possessed the downstream sequence of Sequence ID No.1 of the WSS1 gene to generate constructs pNA1684 and pNA1686, respectively (Figure 1(b)(A)). Similarly, two RAD51 deletion constructs pNA1722 and pNA1723 were generated by using primer pairs of NAP711 of Sequence ID No. 12 and NAP712 of Sequence ID No.13 for downstream fragment of Sequence ID No. 4; and forward primer NAP708 of Sequence ID No. 14 with reverse primers NAP709 of Sequence ID No. 15 and NAP710 of Sequence ID no.16 for upstream sequences of Sequence ID No. 5 and Sequence ID No.6 (figure 1(b)(B). The two different length upstream sequences and construction of the two constructs for each target gene ensures that both the alleles of the gene are deleted in the genetically modified strain.

[0040] The following PCR condition was used for the amplification of the upstream and downstream fragments of RAD51 and WSS1 genes by Taq DNA polymerases in a standard 100?L PCR reaction.
Initial denaturation- 95°C for 1 min.
Denaturation-95°C for 15 sec.
Annealing-52°C for 30 Sec.
Extension-72°C for 30 sec.
Final Extension-72°C for 3 min.
No. of Cycles-35
[0041] In these constructs, the SAT antibiotic marker is flanked by upstream and downstream gene sequences of RAD51 and WSS1 genes (Fig 1).

Example II

[0042] C. albicans transformation and construction of genetically engineered C. albicans knock out strain
[0043] Using the plasmid constructs in Example I, first a homozygous deletion of WSS1 gene was obtained and then both the alleles of RAD51 gene was deleted to generate double homozygous deletant CNA94 strain. A flow chart describing the gene deletion strategy is given (Fig. 2). Briefly, the KpnI-SacI digested linear DNA fragments from the respective SAT1-cassettes were transformed into wild type Sc5314 C. albicans strain by a modified lithium acetate method at different stages to obtain homozygous deletion of both the genes (4).

[0044] The methodology used for DNA transformation to Candida albicans is described. A single colony of C. albicans was inoculated in 10ml YPD and grown overnight at 300C 180 rpm followed by inoculation of 50ml YPD medium with the overnight grown culture (1%) and grow at 30 0C until the O.D.600 reaches at 0.8-1.0. This is followed by spinning at 4000 rpm for 5 min at room temperature to pellet down the cells, Thereafter the pellet is washed in 20 ml sterile water followed by LiOAc/TE solution. The pellet is resuspended in 3 volumes of the pellet in LiOAc/TE solution (approx 300?l). The cell suspension is then aliquot in 100?l aliquots for each transformation and the transforming DNA is added. 700?l of PEG solution (filter sterilized) is added and the mixture is incubated at 300C overnight (12-14hrs). Further, heat shock is provided at 420C for 45 min. The pellets were obtained by centrifuging the cells at 4000 rpm for 5min at room temperature. PEG solution is discarded and the pellet is re-suspended in 1ml YPDU (3hour) at 300 C. This is then centrifuged at 3000rpm for 5min at room temperature. The pellet is resuspended in 100ul of 1X TE buffer (sterile) and plate on YPDU+agar plates with nourseothricin antibiotic (100?g/ml) selection marker. The plates are incubated at 300C for 2-3 days to obtain the transformants.

[0045] The methodology used for homozygous gene deletion in Candida albicans is described briefly. About 10?g of deletion constructs (pNA1686 / pNA1684 / pNA1722 / pNA1723) was digested with 5 units each of KpnI-SacI restriction enzymes at 37 ?C for 6 hrs. Total reaction was resolved in agarose gel electrophoresis and two fragments (~5 kb and 3Kb) were obtained. Only the 5Kb fragment that contains the upstream and downstream sequence of a specific gene with the SAT1 flipper cassette was eluted by using GeneJet Gel Extraction kit from ThermoFisher for transformation to C. albicans as described above. A flowchart of transformation of four different DNA fragments at different time points have been given (Fig. 2). First, the fragment from shorter upstream deletion construct (pNA1686) was transformed into Candida cells and selected the transformants on YPD+ nourseothricin (NAT) plate. The transformed colonies were screened for integration of deletion cassette to the targeted locus by colony PCR using respective gene specific upstream forward and NAP336 reverse primers. NAP336 of Sequence ID No. 17 (5’-GAA ATC CAG ACA GTC GAG-3’) hybridizes near the MAL2 promoter of the pSFS2 vector. The positive colonies showing the integration of deletion cassette were inoculated in 2ml of 5% YPM for 3-5 days for curing of cassette. (Maltose should be < 1 week old). Maltose induces the expression of FLIP recombinase to remove the SAT1 sequence from the genome leaving behind the FRT sequence in the integrated locus. This will facilitate curing of the NAT marker, so that fresh round of transformation can be done with other SAT1 cassettes. The curing of deletion cassette in strains were further confirmed by streaking on YPD media without and with NAT presence. Then colony PCR with ORF specific primers were performed to check deletion. Presence of two PCR products of (one corresponding to full-length and second smaller one for deletant) ensured heterozygous deletion strain. To this heterozygous deletion strain of C. albicans fragment of longer upstream deletion cassette (pNA1684) was transformed and steps from Screen the transformed colonies for integration of deletion cassette to the targeted locus by colony PCR to confirmation of deletion by colony PCR were followed to obtain WSS1 homozygous deletion. The following PCR condition was used for the confirmation of RAD51 and WSS1 genes knockouts by Taq DNA polymerases in a standard 50?L PCR reaction. Initial denaturation- 95°C for 1 min, Denaturation-95°C for 15 sec, Annealing-52°C for 30 Sec, Extension-72°C for 1 min, Final Extension-72°C for 3 min, No. of Cycles-35 The primer sets used for the confirmation of wss1?? are – NAP541 of Sequence ID No. 9 and NAP545 of Sequence ID No. 8. The primer sets used for the confirmation of rad51?? are – NAP708 of Sequence ID No. 14 and NAP712 of Sequence ID No.13. Using similar procedure RAD51 gene was deleted to get CNA94.
[0046] In conclusion, Maltose inducible Flip-recombinase facilitates recycling of SAT1 cassettes; and two-steps recycling by two different knockout constructs of same gene prompts efficient deletion. In each steps various gene deletions were confirmed by gene specific primers and DNA sequencing.

Example III

Growth Rate analysis of rad51?wss1? (CNA94) strain
[0047] The growth rates of the wild type and rad51?wss1? (CNA94) in liquid YPD media by measuring the absorbance of the cultures at 600 nm at an interval of 2 hours for 16 hours (Fig 3A). The growth curve analyses suggested that CNA94 grows extremely slower than the wild type strain (Fig. 3B). Spot analyses further suggested that CNA94 is both cold and high temperature sensitive. Comparison to wild type, rad51?wss1? cells are also hypersensitive to genotoxic agents such as hydroxyurea (HU), cisplatin, formaldehyde, UV radiation etc. Pulse field gel electrophoresis analysis of their total chromosomal DNA suggested that CNA94 exhibits aneuploidy as chromosome # 6 is either absent or migrate abnormally (Fig. 3C).

Example IV

Morphological analysis
[0048] A number of determinants both from the host and fungus decide whether the Candida cells to remain as a commensals or pathogens, although not a single component has yet conclusively shown to be essential. The most appreciated important virulence factor of C. albicans is linked to morphological transition. Normally blastoconidia (round yeast cells) are exclusively found in immunocompetent individuals, however, a mixture of blastoconidia, pseudohyphae, and hyphae forms are frequently found in infected patients. Thus, strains of C. albicans that undergo morphological transitions have been shown to be pathogenic than those that locked in any of the morphological structures. Interestingly, while the wild type cells are oval in shaped, rad51?wss1? cells are filamentous. When the wild type cells undergo morphological switching to pseudohyphae from oval shaped in the presence of HU, MMS, TBHP, and cisplatin, CNA94 remained pseudohyphal. Serum and spider media induces filamentation in wild type cell. While wild type C. albicans cells switched their morphology in serum and spider media, CNA95 was locked in filamentation structures (Fig. 4 A, B, and C).

Example V

Pathogenicity of rad51?wss1? (CNA94) strain
[0049] The intravenous challenge of C. albicans in mouse models is a routinely used technique to study systemic disseminated infections (3). In this challenged experimental model, animals die within a week of inoculation due to severe sepsis in most of the organs with the highest fungal load in kidneys, the phenotype mimics the severe clinical cases. To determine the virulence and pathogenicity of CNA94 strain of C. albicans, BLAB/C male mice (n-8 x 2) were injected with a fungal dose of 5 x 105 CFU per mouse via the lateral tail vein, and monitored for the mice, which succumbed to infection over a period of 30 days. While all animals succumbed to death within 13 days of inoculation in wild type C. albicans injected group, 100 % of mice survived upon rad51?wss1? strain inoculated group (Fig. 5 A, B, and C).. The rad51?wss1? strain injected group of mice survived similar to saline control injection. Microscopic examination of PAS stained autopsy of kidney sections obtained from wild type infected BALB/c mice revealed the presence of C. albicans cells in both cortex and medulla of the kidney, and similarly, CFU of C. albicans cells was estimated to be very high in kidneys. Thus, we claimed that CNA94 is an avirulent strain of C. albicans and could be explored further to develop live attenuated whole cell vaccine.

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[0052] The crux of the invention is claimed implicitly and explicitly through the following claims.