Commentary - Journal of AIDS and HIV Treatment (2021) Volume 3, Issue 1
Dihydropteroate Synthase (DHPS) Gene Mutations in Human Pneumocystosis
Bijay Ranjan Mirdha*
All India Institute of Medical Sciences, Department of Microbiology, New Delhi 110029, India
- *Corresponding Author:
- Bijay Ranjan Mirdha;
E-mail: firstname.lastname@example.org; mirdhabr2078gmail.com
Received date: January 21, 2021; Accepted date: March 15, 2021
Citation: Mirdha BR. Dihydropteroate Synthase (DHPS) Gene Mutations in Human Pneumocystosis. J AIDS HIV Treat. 2021;
Copyright: © 2021 Mirdha BR. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Extraordinary journey began on June 18, 1981, the Centre for Disease Control and Prevention (CDC) in United States reported a cluster of Pneumocystis carinii pneumonia (now known as Pneumocystis jirovecii (P. jirovecii) pneumonia or Pneumocystis pneumonia in five gay men in Los Angeles. It was known as Gay Related Immune Deficiency (GRID). Subsequently, it was confirmed that about 50% of people with the syndromes were not gay, hence, the name was changed to acquired immune deficiency Syndrome (AIDS). Following Human immunodeficiency virus infection (HIV) and AIDS, substantial increase in the number of cases of Pneumocystis pneumonia were reported throughout the world. Two seemingly related events emerged over the years in human pneumocystosis, one relates to the increasing use of prophylaxis in infected individuals and the other about drug resistance.
Despite decades of research in drug development trimethoprim and sulfamethoxazole (TMP-SMZ), a mixture of 1:5 ratio, remains the most effective first line regimen for both anti-Pneumocystis prophylaxis and therapy since 1990s. Anti-Pneumocystis activity of TMPSMZ is however, almost entirely to sulfamethoxazole . Many sulfa drugs (short, intermediate and long acting) are close structural analogs of para-aminobenzoic acid (pABA), that exhibit its antimicrobial activities by competing with pABA at the dihydropteroate synthase (DHPS) active site. This leads to critical depletion of cellular folate levels [2,3]. Folates being the crucial cofactors in the synthesis of thymidine, purines, certain amino acids and pantothenic acid, generally play critical roles in one-carbon transfer reactions in cell metabolism. Higher eukaryotes obtain folic acid and reduced folate polyglutamates from dietary sources by uptake through membrane-associated folate transport proteins. In contrast, prokaryotes and some lower eukaryotes are obligatory synthesizer of folate de novo . DHPS and dihydrofolate reductase (DHFR) are the two key enzymes that are necessary for P. jirovecii to synthesize folate. These two represent the enzymatic targets for the antifolate based treatment with TMP-SMZ. In P. jirovecii, DHPS is part of a tri-functional protein along with two other enzymes dihydroneopterin aldolase and hydroxymethyldihydropterin pyrophosphokinase, essential in the folic acid biosynthesis pathway. In their study, Volpe et al. demonstrated that Pneumocystis contains DHPS activity using a new high performance liquid chromatography (HPLC) based assay. It was also observed in their study that the effects of various sulfa drugs on the DHPS were quite different than those of the Escherichia coli DHPS. For example, the inhibitory constant (Ki) for
different organisms were different, reflecting the fact
that fundamental differences in the enzyme-active site
structures differs between genus and species .
Besides concerns about the toxicity of the TMP-SMZ treatment and low tolerance to sulfa-based drugs in some patients, there are growing evidences of the emerging resistance of the P. jirovecii with the acquired mutations in the targeted enzymes. Earlier studies reported about sequence variants in both DHPS and DHFR of P. jirovecii, suggesting the development of resistance upon exposure to the drug. Subsequently, as the number of Pneumocystis pneumonia (PCP) patients unresponsive to TMP-SMZ increased over the years and the corresponding strains of the pathogen were sequenced, it became possible to draw statistically significant associations to estimate possible risks of resistance upon prior exposure to the drug [7,8].
A growing number of reports identified DHPS mutations primarily at codon 55 and 57 which were associated with higher rates of treatment failure in PCP patients treated with intravenous TMP-SMZ [7,9,10]. These mutations change a single nucleotide in a base triplet. The most frequent nonsynonymous single nucleotide polymorphisms (SNPs) in P. jirovecii DHPS have been observed at positions 165 and 171, the combination of which have led to four different possible genetic alleles. Most common non-synonymous mutations single nucleotide polymorphisms (SNPs) in the P. jirovecii DHPS gene are located at nucleotide positions
165 (A–G) and 171 (C–T), causing amino acid substitutions
at codon 55 (Thr to Ala) and 57 (Pro to Ser) in a highly
conserved region of one of the putative active sites of
the enzyme [7,11]. The presence of the most common
DHPS mutations at codon 55 (Thr55Ala) and codon 57
(Pro57Ser) may be a cause of drug resistance . The four
different DHPS genotypes are WW (wild type in positions
165 and 171), WM (wild type in 165 and mutated in 171),
MW (mutated in position 165 and wild type in 171) and
MM (mutated in positions 165 and 171). Haplotypes for
DHPS have been assigned based upon codons 55 and 57.
As the study of drug efficacy cannot be directly assessed in the absence of a reliable P. jirovecii cultivation system, use of molecular method is the only possible way of studying the prevalence of drug resistance. Interestingly, there are large geographic variations in the frequency of DHPS mutations identified in patient samples throughout the world. Several studies in high income settings have shown relatively higher mutations. Reported prevalence of DHPS codon 55 or 57 mutations was as high as 69% in the USA, whereas in European countries, the prevalence ranged from 0 to 36% [13,14]. In one of the initial USAmerican study, 22 out of 29 samples were positive for DHPS mutations after drug exposure , in contrast, two German studies showed much lower prevalence. In addition, occurrence of identical genotypes in these two German studies indicated stable transmission of few P. jirovecii strains with little genetic variation over time and
Correlation between mutation(s) in the P. jirovecii DHPS gene and resistance to SMZ have been reported by various studies [9,18-20]. P. jirovecii DHPS with the double mutations was associated with lower susceptibility to SMZ. Furthermore, double mutations resulted in threefold increase in minimum inhibitory concentrations (MICs) to that of the wild-type DHPS . In Europe, the prevalence of the double mutated genotypes has been low (<3%), compared to 40% that has been reported in USA. A threefold increase in mortality rate has also been reported in patients with DHPS mutation, than those infected with wild type. Moreover, double mutated allele has been the predominant P. jirovecii DHPS genotype found in patients experiencing sulfa-drug prophylaxis failure , suggesting the existence of a selective pressure. However, some studies did not find any link between DHPS mutations and treatment failure.
Reports from low-and middle-income settings, where the burdens of AIDS and PCP are greatest, DHPS mutation in P. jirovecii have shown variable results. Prevalence of PCP amongst HIV-infected patients with pneumonia was 27% in some of the African countries . In a cross-sectional study, HIV-infected Ugandans had low prevalence of Pneumocystis pneumonia, however, all P. jirovecii isolates harbored mutations in the DHPS gene . In this study
by Taylor et al., most patients individually denied prior
exposure to prophylaxis with antifolate drugs. Such high
prevalence of DHPS mutation despite the lack of antifolate
prophylaxis may suggest two nonexclusive processes such
as (i) inter-human transmission of mutant strains or (ii)
population-level selective pressure exerted by antifolate use
for other indications. Inter-human transmission of mutant
strains has been inferred from ecological studies of mutant
genotypes in which geographical residence was associated
with the genotype . Since the first putative description of inter-human transmission of P. jirovecii in 1967 , a
large number of nosocomial outbreaks of PCP (sometimes
referred to as clusters) have been reported in the literature.
In a Korean study, conducted from 2007 to 2013, no DHPS
mutations were observed in any of the episode of PCP in
patients studied . Although significant prevalence of
mutations in DHPS (20–37%) have been reported from
different countries [27,28], only one DHPS mutant was
reported among 52 strains studied in Japan . These
variable findings may be due to geographical differences.
The association between use of TMP-SMZ as prophylaxis
and the frequency of mutations was stronger for the studies
that included multiple isolates than for those that did not.
These differences suggest the possibility that exposure to
multiple courses of sulfa prophylaxis increases the chances
of DHPS mutations. In contrast, low prevalence of DHPS
mutations have been observed in many studies conducted
in Latin American countries with similar usage of sulfa
drugs, suggesting possible additional sources of resistant
genotypes. However, Chile is the only exception, where
prevalence of DHPS mutations was high, presumably
acquired through inter-human transmission.
Kazanjian et al. revealed DHP gene mutation rate of 7% (1/15) in AIDS patients with PCP in Beijing. In their study among non?HIV?infected patients with Pneumocystis pneumonia, between January 2008 and April 2011, mutations at Thr55Ala and Pro57Ser amino acid substitutions were not observed, instead, two other nonsynonymous mutations, i.e Asp90Asn and Glu98Lys were identified from two patients . In a study from India, conducted in 76 HIV-positive patients, 22.4% (17/76) were positive for P. jirovecii. Upon DHPS gene sequencing, a novel nucleotide substitution at position 288 (Val96Ile) was observed in three patients. All three patients infected with this particular mutant genotype had severe episodes of PCP, did not respond to TMP-SMZ treatment and had fatal outcome (P=0.005) .
Finally, what we observe is that although conflicting and variable reports are available pertaining to DHPS mutations in P. jirovecii pneumonia, the mutational surveillance study may have advantages to detect minority variants and allow for the detection of drug resistance to generate evolving scenario that may help to develop alternative treatment algorithm/s.
- Walzer PD, Foy J, Steele P, Kim CK, White M, Klein
RS, et al. Activities of antifolate, antiviral and other drugs
in an immunesuppressed rat model of Pneumocystis
carinii pneumonia. Antimicrob Agents Chemother.
- Achari A, Somers DO, Champness JN, Bryant PK,
Rosemond J, Stammers DK. Crystal structure of the
anti-bacterial sulfonamide drug target dihydropteroate
synthase. Nat Struct Biol. 1997;4(6):490-97.
- Wood DD. The relationship of p-aminobenzoic acid to
the mechanism of the action of sulphanilamide. Br J Exp
- Matherly LH. Molecular and cellular biology of the
human reduced folate carrier. Prog Nucleic Acid Res Mol
- Volpe F, Dyer M, Scaife JG, Darby G, Stammers DK,
Delves, CJ. The multifunctional folic acid synthesis fas gene
of Pneumocystis carinii appears to encode dihydropteroate
synthase and hydroxymethyldihydropterin pyrophos
phokinase. Gene. 1992;112(2):213-18.
- Merali S, Zhang Y, Slona D, Meshnick S. Inhibition of
Pneumocystis carinii dihydropteroate synthetase by sulfa
drugs. Antimicrob Agents Chemother. 1990;34(6):1075-
- Lane BR, Ast JC, Hossler PA, Mindell DP, Bartlett MS,
Smith JW, et al. Dihydropteroate synthase polymorphisms
in Pneumocystis carinii. J Infect Dis. 1997;175(2):482-85.
- Navin TR, Beard CB, Huang, L del Rio C, Lee S,
Pieniazek NJ, et al. Effect of mutations in Pneumocystis
carinii dihydropteroate synthase gene on outcome of P
carinii pneumonia in patients with HIV-1: a prospective
- Helweg-Larsen J, Benfield TL, Eugen-Olsen J,
Lundgren JD, Lundgren B. Effects of mutations in
Pneumocystis carinii dihydropteroate synthase gene on
outcome of AIDS-associated P. carinii pneumonia. Lancet.
- Lee SM, Cho YK, Sung YM, Chung DH, Jeong SH, Park
JW, et al. A Case of Pneumonia Caused by Pneumocystis
jirovecii Resistant to Trimethoprim–Sulfamethoxazole.
Korean J Parasitol. 2015;53(3):321-27.
- Kazanjian P, Locke AB, Hossler PA, Lane BR, Bartlett
MS, Smith JW, et al. Pneumocystis carinii mutations associated with sulfa and sulfone prophylaxis failures in
AIDS patients. AIDS. 1998;12(8):873-78.
- Tyagi AK, Mirdha BR, Luthra K, Guleria R,
Mohan A, Singh UB, et al. Pneumocystis jirovecii
dihydropteroate synthase (DHPS) genotypes in non-HIVimmunocompromised
patients: a tertiary care reference
health centre study. Med Mycol. 2011;49(2):167-71.
- Beard CB, Carter JL, Keely SP, Huang L, Pieniazek NJ,
Moura IN, et al. Genetic variation in Pneumocystis carinii
isolates from different geographic regions: implications
for transmission. Emerg Infect Dis. 2000;6(3):265-72.
- Le Gal S, Robert-Gangneux F, Perrot M, Rouille A,
VirmauxM, Damiani C, et al. Absence of Pneumocystis
dihydropteroate synthase mutants in Brittany France.
Diagn Microbiol Infect Dis. 2013;76(1):113-15.
- Ma L, Borio L, Masur H, Kovacs JA. Pneumocystis
carinii dihydropteroate synthase but not dihydrofolate
reductase gene mutations correlate with prior
trimethoprim–sulfamethoxazole or dapsone use. J Infect
- Riebold D, Fritzsche C, Lademann M, Bier A, Reisinger
EC. Pneumocystis jirovecii dihydropteroate synthase gene
mutations at codon 171 but not at codons 55 or 57 detected
in Germany. Clin Infect Dis. 2006;42(4):582-83.
- Suárez I, Roderus L, van Gumpel E, Jung N, Lehmann
C, Fätkenheuer G, et al. Low prevalence of DHFR and DHPS
mutations in Pneumocystis jirovecii strains obtained from
a German cohort. Infection. 2017;45(3):341-47.
- Ma L, Kovacs JA, Cargnel A, Valerio A, Fantoni G,
Atzori C. Mutations in the dihydropteroate synthase gene
of human-derived Pneumocystis carinii isolates from Italy
are infrequent but correlate with prior sulfa prophylaxis. J
Infect Dis. 2002;185(10):1530-32.
- Visconti E, Ortona E, Margutti P, Marinaci S, Zolfo
M, Mencarini P, et al. Identification of dihydropteroate
(DHPS) gene mutant in Pneumocystis carinii in respiratory
samples of HIV+ patients from 1992 to 1997. J Eukaryot
- Queener SF, Cody V, Pace J, Torkelson P, Gangjee
A. Trimethoprim resistance of dihydrofolate reductase
variants from clinical isolates of Pneumocystis jirovecii.
Antimicrob Agents Chemother. 2013;57(10):4990-98.
- Iliades P, Meshnick SR, Macreadie IG. Mutations
in the Pneumocystis jirovecii DHPS gene confer crossresistance
to sulfa drugs. Antimicrob Agents Chemother.
- Hartung TK, Chimbayo D, van Oosterhout JJ, Chikaonda T, van Doornum GJJ, Claas ECJ, et al. Etiology of suspected pneumonia in adults admitted to a high dependency unit in Blantyre, Malawi. Am J Trop Med Hyg. 2011;85(1):105-12.
- Taylor SM, Meshnick SR, Worodria W, Andama
A, Cattamanchi A, Davis JL, et al. Low prevalence of
Pneumocystis pneumonia (PCP) but high prevalence
of Pneumocystis dihydropteroate synthase (dhps) gene
Mutations in HIV-Infected Persons in Uganda. PLoS ONE.
- Dini L, du Plessis M, Frean J, Fernandez V. High
prevalence of dihydropteroate synthase mutations
in Pneumocystis jirovecii isolated from patients with
Pneumocystis pneumonia in South Africa. J Clin Microbiol.
- Ruskin J, Remington JS. The compromised host
and infection. I. Pneumocystis carinii pneumonia. JAMA.
- Kim T, Lee SO, Hong HL , Lee JY, Kim SH, Choi SH, et
al. Clinical characteristics of hospital-onset Pneumocystis
pneumonia and genotypes of Pneumocystis jirovecii in a single tertiary centre in Korea. BMC Infectious Diseases.
- Gupta R, Mirdha BR, Guleria R, Agarwal SK,
Samantaray JC, Kumar L, et al. Genotypic variation of
Pneumocystis jirovecii isolates in India based on sequence
diversity at mitochondrial large subunit rRNA. Int J Med
- Jarboui MA, Mseddi F, Sellami H, Sellami A, Makni F,
Ayadi A. Genetic diversity of Pneumocystis jirovecii strains
based on sequence variation of different DNA region. Med
- Rabodonirina M, Vaillant L, Taffe P, Nahimana A,
Gillibert RP, Vanhems P, et al. Pneumocystis jirovecii
genotype associated with increased death rate of HIV
infected patients with pneumonia. Emerg Infect Dis.
- Singh Y, Mirdha BR, Guleria R, Kabra SK, Mohan A,
Chaudhry R, et al. Novel dihydropteroate synthase gene
mutation in Pneumocystis jirovecii among HIV-infected
patients in India: putative association with drug resistance
and mortality. J Glob Antimicrob Resist. 2019;17: 236-39.