TY - JOUR
T1 - Lathosterol oxidase (sterol c-5 desaturase) deletion confers resistance to amphotericin b and sensitivity to acidic stress in leishmania major
AU - Ning, Yu
AU - Frankfater, Cheryl
AU - Hsu, Fong Fu
AU - Soares, Rodrigo P.
AU - Cardoso, Camila A.
AU - Nogueira, Paula M.
AU - Lander, Noelia Marina
AU - Docampo, Roberto
AU - Zhang, Kai
N1 - Funding Information:
We thank Hannah Burks (Texas Tech University, Lubbock, Texas, USA) for technical assistance; W. Robert McMaster (University of British Columbia, Vancouver, Canada) and Jay Bangs (University at Buffalo, State University of New York) for kindly providing the anti-GP63 monoclonal antibody 235 and the rabbit anti-T. brucei BiP polyclonal antiserum, respectively; and Catherine Wakeman (Texas Tech University, Lubbock, Texas, USA) for use of the BioTek synergy 4 fluorescence microplate reader. This work was supported by National Institutes of Health grants AI099380 (K.Z.), P30DK020579, P30DK056341, and P41GM103422 (Mass Spectrometry Resource of Washington University). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Publisher Copyright:
© 2020 Ning et al.
PY - 2020/7/1
Y1 - 2020/7/1
N2 - Lathosterol oxidase (LSO) catalyzes the formation of the C-5-C-6 double bond in the synthesis of various types of sterols in mammals, fungi, plants, and protozoa. In Leishmania parasites, mutations in LSO or other sterol biosynthetic genes are associated with amphotericin B resistance. To investigate the biological roles of sterol C-5-C-6 desaturation, we generated an LSO-null mutant line (lso) in Leishmania major, the causative agent for cutaneous leishmaniasis. lso parasites lacked the ergostane-based sterols commonly found in wild-type L. major and instead accumulated equivalent sterol species without the C-5-C-6 double bond. These mutant parasites were replicative in culture and displayed heightened resistance to amphotericin B. However, they survived poorly after reaching the maximal density and were highly vulnerable to the membrane-disrupting detergent Triton X-100. In addition, lso mutants showed defects in regulating intracellular pH and were hypersensitive to acidic conditions. They also had potential alterations in the carbohydrate composition of lipophosphoglycan, a membrane-bound virulence factor in Leishmania. All these defects in lso- were corrected upon the restoration of LSO expression. Together, these findings suggest that the C-5-C-6 double bond is vital for the structure of the sterol core, and while the loss of LSO can lead to amphotericin B resistance, it also makes Leishmania parasites vulnerable to biologically relevant stress. IMPORTANCE Sterols are essential membrane components in eukaryotes, and sterol synthesis inhibitors can have potent effects against pathogenic fungi and trypanosomatids. Understanding the roles of sterols will facilitate the development of new drugs and counter drug resistance. LSO is required for the formation of the C-5-C-6 double bond in the sterol core structure in mammals, fungi, protozoans, plants, and algae. Functions of this C-5-C-6 double bond are not well understood. In this study, we generated and characterized a lathosterol oxidase-null mutant in Leishmania major. Our data suggest that LSO is vital for the structure and membrane-stabilizing functions of leishmanial sterols. In addition, our results imply that while mutations in lathosterol oxidase can confer resistance to amphotericin B, an important antifungal and antiprotozoal agent, the alteration in sterol structure leads to significant defects in stress response that could be exploited for drug development.
AB - Lathosterol oxidase (LSO) catalyzes the formation of the C-5-C-6 double bond in the synthesis of various types of sterols in mammals, fungi, plants, and protozoa. In Leishmania parasites, mutations in LSO or other sterol biosynthetic genes are associated with amphotericin B resistance. To investigate the biological roles of sterol C-5-C-6 desaturation, we generated an LSO-null mutant line (lso) in Leishmania major, the causative agent for cutaneous leishmaniasis. lso parasites lacked the ergostane-based sterols commonly found in wild-type L. major and instead accumulated equivalent sterol species without the C-5-C-6 double bond. These mutant parasites were replicative in culture and displayed heightened resistance to amphotericin B. However, they survived poorly after reaching the maximal density and were highly vulnerable to the membrane-disrupting detergent Triton X-100. In addition, lso mutants showed defects in regulating intracellular pH and were hypersensitive to acidic conditions. They also had potential alterations in the carbohydrate composition of lipophosphoglycan, a membrane-bound virulence factor in Leishmania. All these defects in lso- were corrected upon the restoration of LSO expression. Together, these findings suggest that the C-5-C-6 double bond is vital for the structure of the sterol core, and while the loss of LSO can lead to amphotericin B resistance, it also makes Leishmania parasites vulnerable to biologically relevant stress. IMPORTANCE Sterols are essential membrane components in eukaryotes, and sterol synthesis inhibitors can have potent effects against pathogenic fungi and trypanosomatids. Understanding the roles of sterols will facilitate the development of new drugs and counter drug resistance. LSO is required for the formation of the C-5-C-6 double bond in the sterol core structure in mammals, fungi, protozoans, plants, and algae. Functions of this C-5-C-6 double bond are not well understood. In this study, we generated and characterized a lathosterol oxidase-null mutant in Leishmania major. Our data suggest that LSO is vital for the structure and membrane-stabilizing functions of leishmanial sterols. In addition, our results imply that while mutations in lathosterol oxidase can confer resistance to amphotericin B, an important antifungal and antiprotozoal agent, the alteration in sterol structure leads to significant defects in stress response that could be exploited for drug development.
KW - Amphotericin
KW - Drug resistance mechanisms
KW - Leishmania
KW - Lipid synthesis
KW - Sterols
KW - Stress response
UR - http://www.scopus.com/inward/record.url?scp=85087655730&partnerID=8YFLogxK
U2 - 10.1128/MSPHERE.00380-20
DO - 10.1128/MSPHERE.00380-20
M3 - Article
C2 - 32611698
AN - SCOPUS:85087655730
SN - 2379-5042
VL - 5
JO - mSphere
JF - mSphere
IS - 4
M1 - 00380
ER -