Phosphomannosylation and the functional analysis of the extended Candida albicans MNN4-like gene family

Roberto J. González-Hernández, Kai Jin, Marco J. Hernández-Chávez, Diana F. Diaz-Jimenez, Elías Trujillo-Esquivel, Diana M. Clavijo-Giraldo, Alma K. Tamez-Castrellón, Bernardo Franco, Neil Andrew Robert Gow, Hector Mora-Montes

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Abstract

Phosphomannosylation is a modification of cell wall proteins that occurs in some species of yeast-like organisms, including the human pathogen Candida albicans. These modified mannans confer a negative charge to the wall, which is important for the interactions with phagocytic cells of the immune systems and cationic antimicrobial peptides. In Saccharomyces cerevisiae, the synthesis of phosphomannan relies on two enzymes, the phosphomannosyltransferase Ktr6 and its positive regulator Mnn4. However, in C. albicans, at least three phosphomannosyltransferases, Mnn4, Mnt3 and Mnt5, participate in the addition of phosphomannan. In addition to MNN4, C. albicans has a MNN4-like gene family composed of seven other homologous members that have no known function. Here, using the classical mini-Ura-blaster approach and the new gene knockout CRISPR-Cas9 system for gene disruption, we generated mutants lacking single and multiple genes of the MNN4 family; and demonstrate that, although Mnn4 has a major impact on the phosphomannan content, MNN42 was also required for full protein phosphomannosylation. The reintroduction of MNN41, MNN42, MNN46 or MNN47 in a genetic background lacking MNN4 partially restored the phenotype associated with the mnn4Δ null mutant, suggesting that there is partial redundancy of function between some family members and that the dominant effect of MNN4 over other genes could be due to its relative abundance within the cell. We observed that additional copies of alleles number of any of the other family members, with the exception of MNN46, restored the phosphomannan content in cells lacking both MNT3 and MNT5. We, therefore, suggest that phosphomannosylation is achieved by three groups of proteins: [i] enzymes solely activated by Mnn4, [ii] enzymes activated by the dual action of Mnn4 and any of the products of other MNN4-like genes, with exception of MNN46, and [iii] activation of Mnt3 and Mnt5 by Mnn4 and Mnn46. Therefore, although the MNN4-like genes have the potential to functionally redundant with Mnn4 they apparently do not play a major role in cell wall mannosylation under most in vitro growth conditions. In addition, our phenotypic analyses indicate that several members of this gene family influence the ability of macrophages to phagocytose C. albicans cells.
Original languageEnglish
Article number2156
JournalFrontiers in Microbiology
Volume8
DOIs
Publication statusPublished - 6 Nov 2017

Fingerprint

Candida albicans
Genes
Cell Wall
Enzymes
Clustered Regularly Interspaced Short Palindromic Repeats
Antimicrobial Cationic Peptides
Mannans
Gene Knockout Techniques
Proteins
Aptitude
Phagocytes
Phagocytosis
Saccharomyces cerevisiae
Immune System
Yeasts
Alleles
Macrophages
Phenotype
phosphomannan
Growth

Keywords

  • cell wall
  • Phosphomannosylation
  • Candida albicans
  • Phagocytosis
  • phosphomannosyltransfrase
  • CRISPR-Cas9 system
  • mini-Ura-blaster

Cite this

González-Hernández, R. J., Jin, K., Hernández-Chávez, M. J., Diaz-Jimenez, D. F., Trujillo-Esquivel, E., Clavijo-Giraldo, D. M., ... Mora-Montes, H. (2017). Phosphomannosylation and the functional analysis of the extended Candida albicans MNN4-like gene family. Frontiers in Microbiology, 8, [2156]. https://doi.org/10.3389/fmicb.2017.02156

Phosphomannosylation and the functional analysis of the extended Candida albicans MNN4-like gene family. / González-Hernández, Roberto J.; Jin, Kai; Hernández-Chávez, Marco J.; Diaz-Jimenez, Diana F.; Trujillo-Esquivel, Elías; Clavijo-Giraldo, Diana M.; Tamez-Castrellón, Alma K.; Franco, Bernardo; Gow, Neil Andrew Robert; Mora-Montes, Hector.

In: Frontiers in Microbiology, Vol. 8, 2156, 06.11.2017.

Research output: Contribution to journalArticle

González-Hernández, RJ, Jin, K, Hernández-Chávez, MJ, Diaz-Jimenez, DF, Trujillo-Esquivel, E, Clavijo-Giraldo, DM, Tamez-Castrellón, AK, Franco, B, Gow, NAR & Mora-Montes, H 2017, 'Phosphomannosylation and the functional analysis of the extended Candida albicans MNN4-like gene family' Frontiers in Microbiology, vol. 8, 2156. https://doi.org/10.3389/fmicb.2017.02156
González-Hernández RJ, Jin K, Hernández-Chávez MJ, Diaz-Jimenez DF, Trujillo-Esquivel E, Clavijo-Giraldo DM et al. Phosphomannosylation and the functional analysis of the extended Candida albicans MNN4-like gene family. Frontiers in Microbiology. 2017 Nov 6;8. 2156. https://doi.org/10.3389/fmicb.2017.02156
González-Hernández, Roberto J. ; Jin, Kai ; Hernández-Chávez, Marco J. ; Diaz-Jimenez, Diana F. ; Trujillo-Esquivel, Elías ; Clavijo-Giraldo, Diana M. ; Tamez-Castrellón, Alma K. ; Franco, Bernardo ; Gow, Neil Andrew Robert ; Mora-Montes, Hector. / Phosphomannosylation and the functional analysis of the extended Candida albicans MNN4-like gene family. In: Frontiers in Microbiology. 2017 ; Vol. 8.
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title = "Phosphomannosylation and the functional analysis of the extended Candida albicans MNN4-like gene family",
abstract = "Phosphomannosylation is a modification of cell wall proteins that occurs in some species of yeast-like organisms, including the human pathogen Candida albicans. These modified mannans confer a negative charge to the wall, which is important for the interactions with phagocytic cells of the immune systems and cationic antimicrobial peptides. In Saccharomyces cerevisiae, the synthesis of phosphomannan relies on two enzymes, the phosphomannosyltransferase Ktr6 and its positive regulator Mnn4. However, in C. albicans, at least three phosphomannosyltransferases, Mnn4, Mnt3 and Mnt5, participate in the addition of phosphomannan. In addition to MNN4, C. albicans has a MNN4-like gene family composed of seven other homologous members that have no known function. Here, using the classical mini-Ura-blaster approach and the new gene knockout CRISPR-Cas9 system for gene disruption, we generated mutants lacking single and multiple genes of the MNN4 family; and demonstrate that, although Mnn4 has a major impact on the phosphomannan content, MNN42 was also required for full protein phosphomannosylation. The reintroduction of MNN41, MNN42, MNN46 or MNN47 in a genetic background lacking MNN4 partially restored the phenotype associated with the mnn4Δ null mutant, suggesting that there is partial redundancy of function between some family members and that the dominant effect of MNN4 over other genes could be due to its relative abundance within the cell. We observed that additional copies of alleles number of any of the other family members, with the exception of MNN46, restored the phosphomannan content in cells lacking both MNT3 and MNT5. We, therefore, suggest that phosphomannosylation is achieved by three groups of proteins: [i] enzymes solely activated by Mnn4, [ii] enzymes activated by the dual action of Mnn4 and any of the products of other MNN4-like genes, with exception of MNN46, and [iii] activation of Mnt3 and Mnt5 by Mnn4 and Mnn46. Therefore, although the MNN4-like genes have the potential to functionally redundant with Mnn4 they apparently do not play a major role in cell wall mannosylation under most in vitro growth conditions. In addition, our phenotypic analyses indicate that several members of this gene family influence the ability of macrophages to phagocytose C. albicans cells.",
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author = "Gonz{\'a}lez-Hern{\'a}ndez, {Roberto J.} and Kai Jin and Hern{\'a}ndez-Ch{\'a}vez, {Marco J.} and Diaz-Jimenez, {Diana F.} and El{\'i}as Trujillo-Esquivel and Clavijo-Giraldo, {Diana M.} and Tamez-Castrell{\'o}n, {Alma K.} and Bernardo Franco and Gow, {Neil Andrew Robert} and Hector Mora-Montes",
note = "We thank Luz A. L{\'o}pez-Ram{\'i}rez (Universidad de Guanajuato) for technical assistance. This work was supported by Consejo Nacional de Ciencia y Tecnolog{\'i}a (ref. CB2011/166860; PDCPN2014-247109, and FC 2015-02-834), Universidad de Guanajuato (ref. 000025/11; 0087/13; ref. 1025/2016; Convocatoria Institucional para Fortalecer la Excelencia Acad{\'e}mica 2015; CIFOREA 89/2016), Programa de Mejoramiento de Profesorado (ref. UGTO-PTC-261), and Red Tem{\'a}tica Glicociencia en Salud (CONACYT-M{\'e}xico). NG acknowledges the Wellcome Trust (086827, 075470, 101873, and 200208) and MRC Centre for Medical Mycology for funding (N006364/1). KJ was supported by a research visitor grant to Aberdeen from China Scholarship Council (CSC No. 201406055024). The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2017.02156/full#supplementary-material",
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T1 - Phosphomannosylation and the functional analysis of the extended Candida albicans MNN4-like gene family

AU - González-Hernández, Roberto J.

AU - Jin, Kai

AU - Hernández-Chávez, Marco J.

AU - Diaz-Jimenez, Diana F.

AU - Trujillo-Esquivel, Elías

AU - Clavijo-Giraldo, Diana M.

AU - Tamez-Castrellón, Alma K.

AU - Franco, Bernardo

AU - Gow, Neil Andrew Robert

AU - Mora-Montes, Hector

N1 - We thank Luz A. López-Ramírez (Universidad de Guanajuato) for technical assistance. This work was supported by Consejo Nacional de Ciencia y Tecnología (ref. CB2011/166860; PDCPN2014-247109, and FC 2015-02-834), Universidad de Guanajuato (ref. 000025/11; 0087/13; ref. 1025/2016; Convocatoria Institucional para Fortalecer la Excelencia Académica 2015; CIFOREA 89/2016), Programa de Mejoramiento de Profesorado (ref. UGTO-PTC-261), and Red Temática Glicociencia en Salud (CONACYT-México). NG acknowledges the Wellcome Trust (086827, 075470, 101873, and 200208) and MRC Centre for Medical Mycology for funding (N006364/1). KJ was supported by a research visitor grant to Aberdeen from China Scholarship Council (CSC No. 201406055024). The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2017.02156/full#supplementary-material

PY - 2017/11/6

Y1 - 2017/11/6

N2 - Phosphomannosylation is a modification of cell wall proteins that occurs in some species of yeast-like organisms, including the human pathogen Candida albicans. These modified mannans confer a negative charge to the wall, which is important for the interactions with phagocytic cells of the immune systems and cationic antimicrobial peptides. In Saccharomyces cerevisiae, the synthesis of phosphomannan relies on two enzymes, the phosphomannosyltransferase Ktr6 and its positive regulator Mnn4. However, in C. albicans, at least three phosphomannosyltransferases, Mnn4, Mnt3 and Mnt5, participate in the addition of phosphomannan. In addition to MNN4, C. albicans has a MNN4-like gene family composed of seven other homologous members that have no known function. Here, using the classical mini-Ura-blaster approach and the new gene knockout CRISPR-Cas9 system for gene disruption, we generated mutants lacking single and multiple genes of the MNN4 family; and demonstrate that, although Mnn4 has a major impact on the phosphomannan content, MNN42 was also required for full protein phosphomannosylation. The reintroduction of MNN41, MNN42, MNN46 or MNN47 in a genetic background lacking MNN4 partially restored the phenotype associated with the mnn4Δ null mutant, suggesting that there is partial redundancy of function between some family members and that the dominant effect of MNN4 over other genes could be due to its relative abundance within the cell. We observed that additional copies of alleles number of any of the other family members, with the exception of MNN46, restored the phosphomannan content in cells lacking both MNT3 and MNT5. We, therefore, suggest that phosphomannosylation is achieved by three groups of proteins: [i] enzymes solely activated by Mnn4, [ii] enzymes activated by the dual action of Mnn4 and any of the products of other MNN4-like genes, with exception of MNN46, and [iii] activation of Mnt3 and Mnt5 by Mnn4 and Mnn46. Therefore, although the MNN4-like genes have the potential to functionally redundant with Mnn4 they apparently do not play a major role in cell wall mannosylation under most in vitro growth conditions. In addition, our phenotypic analyses indicate that several members of this gene family influence the ability of macrophages to phagocytose C. albicans cells.

AB - Phosphomannosylation is a modification of cell wall proteins that occurs in some species of yeast-like organisms, including the human pathogen Candida albicans. These modified mannans confer a negative charge to the wall, which is important for the interactions with phagocytic cells of the immune systems and cationic antimicrobial peptides. In Saccharomyces cerevisiae, the synthesis of phosphomannan relies on two enzymes, the phosphomannosyltransferase Ktr6 and its positive regulator Mnn4. However, in C. albicans, at least three phosphomannosyltransferases, Mnn4, Mnt3 and Mnt5, participate in the addition of phosphomannan. In addition to MNN4, C. albicans has a MNN4-like gene family composed of seven other homologous members that have no known function. Here, using the classical mini-Ura-blaster approach and the new gene knockout CRISPR-Cas9 system for gene disruption, we generated mutants lacking single and multiple genes of the MNN4 family; and demonstrate that, although Mnn4 has a major impact on the phosphomannan content, MNN42 was also required for full protein phosphomannosylation. The reintroduction of MNN41, MNN42, MNN46 or MNN47 in a genetic background lacking MNN4 partially restored the phenotype associated with the mnn4Δ null mutant, suggesting that there is partial redundancy of function between some family members and that the dominant effect of MNN4 over other genes could be due to its relative abundance within the cell. We observed that additional copies of alleles number of any of the other family members, with the exception of MNN46, restored the phosphomannan content in cells lacking both MNT3 and MNT5. We, therefore, suggest that phosphomannosylation is achieved by three groups of proteins: [i] enzymes solely activated by Mnn4, [ii] enzymes activated by the dual action of Mnn4 and any of the products of other MNN4-like genes, with exception of MNN46, and [iii] activation of Mnt3 and Mnt5 by Mnn4 and Mnn46. Therefore, although the MNN4-like genes have the potential to functionally redundant with Mnn4 they apparently do not play a major role in cell wall mannosylation under most in vitro growth conditions. In addition, our phenotypic analyses indicate that several members of this gene family influence the ability of macrophages to phagocytose C. albicans cells.

KW - cell wall

KW - Phosphomannosylation

KW - Candida albicans

KW - Phagocytosis

KW - phosphomannosyltransfrase

KW - CRISPR-Cas9 system

KW - mini-Ura-blaster

U2 - 10.3389/fmicb.2017.02156

DO - 10.3389/fmicb.2017.02156

M3 - Article

VL - 8

JO - Frontiers in Microbiology

JF - Frontiers in Microbiology

SN - 1664-302X

M1 - 2156

ER -