Widespread genomic heterogeneity at the type II NAD(P)H dehydrogenase locus predisposes Cryptosporidium to clofazimine resistance

Widespread genomic heterogeneity at the type II NAD(P)H dehydrogenase locus predisposes Cryptosporidium to clofazimine resistance | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Widespread genomic heterogeneity at the type II NAD(P)H dehydrogenase locus predisposes Cryptosporidium to clofazimine resistance View ORCID Profile Gracyn Y. Buenconsejo , View ORCID Profile Sebastian Shaw , View ORCID Profile Rui Xiao , View ORCID Profile Aurelia Balestra , View ORCID Profile Keenan M. O’Dea , View ORCID Profile Peng Jiang , Bingjie Xu , Dongqiang Wang , View ORCID Profile Guan Zhu , View ORCID Profile Daniel P. Beiting , View ORCID Profile Boris Striepen doi: https://doi.org/10.1101/2025.10.07.680968 Gracyn Y. Buenconsejo 1 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania , Philadelphia, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gracyn Y. Buenconsejo Sebastian Shaw 1 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania , Philadelphia, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sebastian Shaw Rui Xiao 1 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania , Philadelphia, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rui Xiao Aurelia Balestra 1 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania , Philadelphia, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Aurelia Balestra Keenan M. O’Dea 1 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania , Philadelphia, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Keenan M. O’Dea Peng Jiang 2 State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University , Changchun, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Peng Jiang Bingjie Xu 2 State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University , Changchun, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dongqiang Wang 2 State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University , Changchun, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Guan Zhu 2 State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University , Changchun, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Guan Zhu Daniel P. Beiting 1 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania , Philadelphia, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Daniel P. Beiting Boris Striepen 1 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania , Philadelphia, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Boris Striepen For correspondence: striepen{at}upenn.edu Abstract Full Text Info/History Metrics Preview PDF Abstract The parasite Cryptosporidium is a leading cause of life-threatening diarrheal disease, and effective treatment is not available. The discovery of potent anti- Cryptosporidium activity of clofazimine, offered the opportunity to repurpose a drug already used to treat leprosy and tuberculosis. However, clofazimine failed in a human trial, which was attributed to poor bioavailability. Here, we observed differential susceptibility among parasite isolates which we exploit to map the mode of action to type II NADH dehydrogenase (NDH2) in an unbiased genetic cross. Targeted genetic ablation of NDH2 resulted in profound clofazimine resistance, and biochemical studies demonstrated NDH2 mediated electron transfer to clofazimine. Through genomic analyses we uncovered heterogeneity at the NDH2 locus for C. parvum and C. hominis and widespread carriage of a conserved attenuated allele across multiple continents. This heterogeneity allows parasites genomically linked through frequent sexual recombination to adjust to changing NDH2 requirements and predisposes Cryptosporidium to evade clofazimine treatment. Introduction The apicomplexan parasite Cryptosporidium is an important cause of intestinal disease in a variety of epidemiological settings. The transmissive oocyst stage is inherently resistant to water chlorination and waterborne outbreaks occur regularly in the United States despite advanced water treatment in place. Cryptosporidium has long been recognized as an life-threatening opportunistic infection in HIV/AIDS patients, causing watery diarrhea and wasting associated with poor prognosis 1 . Various conditions resulting in reduced cellular immune function including certain cancers, solid organ transplantation and the accompanying immunosuppressive therapy, and multiple primary genetic defects similarly predispose to life-threatening cryptosporidiosis 2 . Despite decades of effort, effective treatment is still unavailable and the clinical management of cryptosporidiosis remains very difficult in these patients 3 . More recently Cryptosporidium was also identified as a leading global cause of severe diarrhea and associated deaths in immunocompetent young children, particularly those experiencing malnutrition 4 . In a vicious circle, cryptosporidiosis itself predisposes children to malnutrition and stunting 5 . In the absence of a vaccine, effective drugs are urgently needed for the treatment of this large pediatric population as well 6 – 8 . Motivated by this need academic and industry laboratories conducted multiple large-scale screens to identify new anti-parasitic compounds 6 , 9 . Acknowledging the economic challenge of drug development for a largely resource poor target population, several of these efforts attempted to leverage prior investments through repurposing of established drugs, screening leads, or cherry picked compound libraries 10 – 12 often focused on antimalarials. Among the findings of these studies was the discovery of potent anti- Cryptosporidium activity of clofazimine 11 , which was widely welcomed, as this drug is already in clinical use for decades and is relatively inexpensive to produce. Clofazimine is a riminophenazine derivative ( Fig. 1a ) developed in the 1950s for the treatment of tuberculosis that was initially sidelined in favor of more potent antibiotics 13 . However, the drug has been in widespread use for the treatment of leprosy, where it is used routinely in combination therapy with rifampin and dapsone 14 . Recently multi-drug resistance has spurred a renewed interest in clofazimine for treating tuberculosis. Following clinical trials 15 the WHO now suggests the use of a 6-month treatment regimen composed of bedaquiline, delamanid, linezolid, levofloxacin, and clofazimine in multidrug-resistant/rifampicin-resistant tuberculosis patients with or without fluoroquinolone resistance 16 . Important questions remain in the tuberculosis field about clofazimine’s safety and side effects, its potential for antimicrobial resistance, and related to both of these questions is the lack of conclusive understanding of its mode of action 17 . The molecule is highly lipophilic and therefore thought to exert its activity at the level of cellular membranes. Among the mechanisms that are debated are: binding to bacterial DNA and inhibiting replication 18 , interference with bacterial redox and energy balance due to competition with menaquinone as an electron acceptor for type 2 NAD(P)H dehydrogenase (NDH2) which is part of the bacterial respiratory chain 19 , 20 , or interference with bacterial potassium transport either directly or mediated through phospholipid metabolism 21 – 23 . Clofazimine resistance has been reported clinically and in experimental selection in Mycobacterium tuberculosis , but without clear genetic clues as to the target and mode of action of the drug 17 . Download figure Open in new tab Figure 1: A genetic cross of C. parvum strains differing in clofazimine (CFZ) susceptibility ( a ) Chemical structure of clofazimine. Tangerine stars indicate atoms positioned to accept electrons during enzymatic reduction. ( b and c ) Parasite growth in HCT-8 tissue culture assessed by measuring luminescence in the presence of the indicated concentrations of clofazimine. Data represent mean and SD of n=5 biological replicates. ( d ) Experimental set up of genetic cross between BG Iowa II (CFZ-resistant) and KVI (CFZ-susceptible). Recombinant progeny was used to infect mice treated with vehicle or escalating doses of CFZ. Oocysts were sampled throughout the selection for IC 50 determination and genome sequencing. ( e ) HCT-8 culture clofazimine susceptibility assay of cross progeny treated with vehicle or drug (mean and SD of n=5 biological replicates). ( f and g ) Parasite burden was measured by following fecal luciferase activity for progeny treated with vehicle ( f ) or clofazimine ( g ); n = 3 mice per cross. Gray boxes indicate treatment windows. Orange boxes indicate times oocyst were collected. Most disappointingly, clofazimine failed in human trial for the treatment of cryptosporidiosis. The trial was designed as a randomized, double-blinded, placebo-controlled study in HIV infected adults suffering from cryptosporidiosis 24 . This was a difficult study, challenged by the very poor health of the participants, difficulties in recruitment, and a resulting lack of randomization. Beyond the lack of efficacy, the study also observed lower than anticipated serum levels for clofazimine in the Cryptosporidium infected treatment group, which was attributed to the severity of diarrheal diseases in those participants. Further studies let the authors to conclude that lower than required bioavailability of the drug 25 , 26 was likely responsible for the observed lack of efficacy. Using a combination of forward and reverse genetic experimentation, we map clofazimine sensitivity of Cryptosporidium to its NDH2 gene and population genomic studies reveal widespread genomic heterogenicity at this specific locus in both C. parvum and C. hominis genomes across the globe. These genetic findings have important consequences for the rapid emergence of resistance and provide additional clues to the interpretation of the clinical failure of clofazimine for the treatment of cryptosporidiosis. They also suggest an unconventional role of NDH2 outside of the mitochondrial respiratory chain, one that appears to benefit from the ability to maintain and modulate a pseudo-diploid stage in a haploid organism through frequent sexual recombination. Results Clofazimine susceptibility varies across different C. parvum strains Clofazimine failed in human trial despite preclinical promise, and this has been linked to poor bioavailability of the drug in patients with severe diarrhea 25 , 26 . However, the initial report describing the anti- Cryptosporidium activity of clofazimine noted lower potency against C. hominis in culture assays. These differences in clofazimine susceptibility as well as preliminary observations on two C. parvum strains, led us to consider the possibility of strain-specific, heritable differences in clofazimine susceptibility. To test this more rigorously we established the half-maximal inhibitory concentration (IC 50 ) for clofazimine for multiple different C. parvum strains. We used ST Iowa II, originally obtained from the Sterling laboratory at the University of Arizona and used in the original drug screen, BG Iowa II (a closely related strain propagated by Bunchgrass Farms and widely used for laboratory experiments), and INRA initially isolated in France. All three are IIa genotypes derived from cattle, in contrast, KVI is a IId strain recently isolated from an infected lamb in Israel 27 . All four strains were engineered to express nanoluciferase, and we measured parasite growth over 48h in a human ileocecal adenocarcinoma (HCT-8) cell culture 28 and performed nine-point dose-response assays ranging from 0.5nM to 5µM with half-log 10 steps ( Fig. 1b , all values normalized to vehicle control for each strain). KVI, INRA and Sterling showed similar susceptibility (IC 50 =3.6nM, 2.1nM and 0.6nM, with 95% confidence intervals (CI) of 2.6 to 4.3, 0.1 to 4.1, and ∞ to 3, respectively) comparable to that initially reported by Love et al., while BG Iowa II was about 10-20 times more resistant (IC 50 =38nM; 95% CI=27 to 52) akin to the earlier measurement for C. hominis 11 . Selecting for clofazimine resistance in a genetic cross We recently developed genetic crosses to study Cryptosporidium 27 , 29 and wondered whether we might be able to exploit this differential susceptibility to map the mechanism of action for clofazimine in this parasite in an unbiased forward genetic fashion. We chose Bunchgrass and KVI as crossing parents as they offer the largest number of distinguishing single nucleotide polymorphisms (SNPs) and validated the susceptibility differential in strains engineered with marker cassettes suitable for a cross (Neo for BG 28 and mutated phenylalanyl tRNA synthase 29 , 30 for KVI, Fig. 1c ). Fig. 1d outlines the design of the cross, interferon-γ knockout ( ifnγ −/− ) mice were infected with both parental parasite strains and treated with BRD7929 and paromomycin to select for recombinant progeny carrying both resistance markers. Oocysts of this progeny were collected and used to infect two new groups of mice, one was treated with escalating doses of clofazimine, the control with vehicle alone, and parasite burden was measured by fecal luciferase for 35 days ( Fig. 1f and g ). This treatment regime transiently repressed parasite growth but did not cure the mice as infection rebounded each time. We collected feces in the recovery periods (orange boxes) following each treatment (gray boxes) with drug or vehicle and isolated oocyst pools. We measured the impact of this selection scheme on clofazimine susceptibility in an in vitro dose-response assays for the final selected progeny pools (derived from days 25-28 post-infection following treatment with 30 mg/kg clofazimine or vehicle). The clofazimine-selected progeny showed marked reduction in susceptibility (IC 50 =35nM; 95% CI=26 to 47) when compared to the vehicle-treated progeny (IC 50 =6nM; 95% CI=5 to 7, Fig. 1e ) suggesting selection for resistance. A single genomic locus is linked to clofazimine susceptibility Genomic DNA was extracted from 5×10 6 oocysts of each of the three pools collected for clofazimine selected and vehicle treated progeny populations, and we carried out high-throughput sequencing to generate robust genome coverage (134-228-fold). Reads were aligned to the C. parvum genome and SNPs were called for the 4700 positions discriminating the parental strains as detailed in the Methods section. Fig. 2a shows allele frequencies across all eight chromosomes compared to the telomere-to-telomere C. parvum BG Iowa II genome 31 , 1 indicates exclusive BG Iowa II and 0 exclusive KVI inheritance. Clofazimine selected populations are shown in red, vehicle in blue, and later timepoints are shown in darker shades. We noted multiple peaks indicating preferred inheritance from one of the parents. These included the loci of the two selectable markers on chromosome 3 and 5, and loci on chromosomes 2, 6, and 7 associated with enhanced virulence and persistence of the KVI parent (please refer to our recent publication 27 for detail on these loci). However, only chromosome 7 showed differences when comparing clofazimine treated and untreated samples ( Fig. 2b , each individual SNP is represented by a dot). In the absence of treatment, KVI alleles dominated due to the virulence locus on this chromosome, upon treatment BG alleles were heavily enriched pointing to preferred inheritance from the more clofazimine resistant parent. We next conducted bulk segregant analysis 27 , 32 to detect and measure genetic linkage. A single narrowly defined quantitative trait locus on chromosome 7 emerged, the statistical support for this locus increased with each round of treatment and dose escalation, with the most highly significant SNP exceeding a final G-value of 300 ( Fig. 2c and d ). Download figure Open in new tab Figure 2: Clofazimine susceptibility is associated with a single locus on chromosome 7 ( a ) Allele frequencies of SNPs distinguishing the parental strains used in the cross for all eight C. parvum chromosomes. Each line represents a progeny pool, those shown in blue were treated with vehicle, those in red with clofazimine. Color shades from light to dark indicate the 3 individual pools that were collected, sequenced and analyzed. ( b ) Allele frequencies of chromosome 7 for the third and final pool collected. Blue = vehicle, red = clofazimine. ( c ) Whole genome G’-values for genetic linkage. Lines are the weighted moving averages for the G′-values, and the significance threshold is shown as an orange dashed line. Dark blue = pool 1, light blue = pool 2 and red = pool 3. ( d ) Highly significant ǪTL on chromosome 7. The G values of pool 3 for each individual SNP are shown as dots, the line is the weighted moving averages for the G′-values, and the significance threshold is shown as an orange dashed line. The 95th percentile of the ǪTL is shown in light blue. Resistance is linked to a two base pair deletion in the type 2 NAD(P)H dehydrogenase gene The highest scoring SNP was found in gene cgd7_1890 resulting in a valine instead of an isoleucine in a KH1 domain-containing putative RNA-binding protein ( Fig. 3a ). This represents a conservative substitution, and while KH1 domain RNA binding proteins can play roles in drug resistance in some cancers 33 , they have not been previously associated with clofazimine resistance. We were thus intrigued to find NDH2 encoded by the next gene downstream of the SNP (cgd7_1900). NDH2 is one of the candidate mechanisms of clofazimine action in Mycobacterium 19 . However, the initial comparison of the published parental genomes showed identical NDH2 sequences in both strains. Our bulk segregant analysis used SNPs to detect ǪTLs. We considered that other variations may impact on drug susceptibility and therefore manually inspected the alignment of raw reads for this section. This revealed a previously unrecognized INDEL, the deletion of two adenines (ΔAA) at positions 81 and 82 of the open reading frame in 100% of all reads from clofazimine-selected parasites, while only 4.5% of all reads from the vehicle-treated parasites showed a deletion at this position. Alignment of the parental genomes detected the ΔAA allele in both parents, with higher frequency in the more resistant strain ( Fig. 3b and 3c ). Download figure Open in new tab Figure 3: Clofazimine selected progeny carry an indel at the 5’ end of the NDH2 gene ( a ) Illumina sequence read alignment of the clofazimine selected third progeny pool. Blue bar in gene cgd7_1890 indicates the SNP with the highest G value identified by bulk segregant analysis. Region highlighted in orange indicates the indel in the NDH2 gene that introduces a frameshift and a premature stop codon at the indicated position. ( b) Indel frequency comparison across the progeny either selected or not selected with clofazimine and the parental strains. ( c ) Ǫuantification of the indel frequency represented in ( b ). Genetic ablation of NHD2 confers high level clofazimine resistance The ΔAA deletion is predicted to result in a frame shift of the NDH2 coding sequence and early termination of the protein after only 47 of its 568 predicted amino acids. We therefore hypothesized resistance to be the consequence of loss of NDH2 activity, rather than a SNP in KH1. To directly test this, we disrupted the NDH2 gene through Cas9 directed insertion of the Nluc/Neo marker in BG Iowa II parasites using paromomycin selection ( Fig. 4a ). Transgenic parasites were readily obtained, and PCR mapping verified appropriate insertion ( Fig. 4b ), and we conclude NDH2 to be dispensable. Next, we compared the sensitivity to clofazimine of the deletion mutant to wildtype BG or KVI parasites in tissue culture growth assays and found the mutant to be highly drug resistant with an IC 50 of 4.1µM ( Fig. 4c ). Based on these findings we reasoned that disruption of the NDH2 locus should be selectable by clofazimine treatment. KVI strain sporozoites were electroporated with a marker-less targeting vector encoding Nluc and tdNeonGreen fluorescent protein along with a plasmid for the expression of Cas9 and a guide RNA targeting NDH2 ( Fig. 4d ). Mice infected with these sporozoites were treated for 7 days with 100 mg/kg/day clofazimine starting 24h post-infection. Activity of the Nluc transgene was detected in the feces of these mice on day four following transfection and continued to rapidly increase ( Fig. 4f ). PCR analysis of genomic DNA demonstrated transgene insertion and complete loss of the wildtype locus ( Fig. 4e ). Oocysts subjected to flow cytometry showed uniformly bright fluorescence and when used to infect HCT-8 cultures all parasite stages were Neon Green positive when observed by fluorescence microscopy ( Fig. 4g and 4h ). We conclude that loss of NDH2 confers resistance to clofazimine, and that clofazimine offers a new selection principle for Cryptosporidium transgenesis. Download figure Open in new tab Figure 4: NDH2 ablation results in high level clofazimine resistance ( a ) Genetic ablation of NDH2 by Cas9 mediated marker insertion. P1 – P4 indicate positions of primers used for PCR mapping ( b ) of the mutant and parental wildtype (WT) strains. ( c ) Clofazimine IC 50 determination in HCT-8 culture of the NDH2 knockout and wildtype strains used in the cross. Data are mean and SD of n=5 biological replicates. ( d ) Strategy and constructs used to test whether NDH2 could be used as a selection marker for C. parvum . P1 – P4 indicate positions of primers used for PCR mapping ( e ) of the resulting transgenics compared to KVI wildtype. ( f ) Luciferase activity of parasites transfected as shown in ( d ) and selected with clofazimine. ( g ) Oocyst of clofazimine selected transgenic were purified from the feces and subjected to flow cytometry (gray: KVI WT; green: KVI tdNeonGreen) or used to infect HCT-8 cells ( h ) and observed by fluorescence microscopy at 48h of culture. Note bright green fluorescence in flow cytometry histogram and micrograph (gray: Hoechst; green: tdNeonGreen; red: VVL). Scale bar, 5 μm. ( i ) Spectrophotometric assay of CpNDH2-WT showing NADH-dependent reduction of menadione (MD), menaquinone-4 (MK4), ubiquinone-2 (CoǪ2), and clofazimine (CFZ). Michaelis–Menten kinetics were observed with MD and MK4, while CoǪ2 and CFZ showed positive cooperativity. Catalytic efficiency ranked MD > MK4 > CoǪ2; CFZ activity was comparable to quinones. ( j ) Kinetic parameters ( V max , K m or K 0.5 , Hill slope) derived from nonlinear regression are summarized in the table. ( k ) Clofazimine inhibits CpNDH2-mediated MD reduction in a dose-dependent manner (relative IC 50 ≈ 2.95 μM; absolute IC 50 > 62.5 μM). The results support competitive reduction of CFZ by CpNDH2. Primers used for genotyping in ( b ) and ( e ) can be found in Supplementary Data 3. Recombinant C. parvum NDH2 recognizes clofazimine as substrate Biochemical studies in bacteria have suggested that clofazimine may compete with natural quinones for electron transfer by NDH2 (the two critical nitrogen positions are highlighted in Fig. 1a ), with subsequent spontaneous reactions giving rise to reactive oxygen species that ultimately damage the cell 19 . To test this for Cryptosporidium , we expressed C. parvum NDH2 in Escherichia coli and purified the recombinant maltose-binding protein fusion in intact and cleaved form (Supplementary Fig.1a). A spectrophotometric assay was established to measure the ability of recombinant enzyme to transfer electrons from NADH to various quinone substrates 34 – 36 . Initial assessment showed that both intact and cleaved forms could catalyze the electron transfer from NADH to menadione with the same efficiency (Supplementary Fig.1b and 1c). We observed activity with menadione (MD), menaquinone-4 (MK4), and ubiquinone-2 (CoǪ2) as substrates, with low-micromolar K m values (or K 0.5 for CoǪ2, which shows positive cooperativity, Fig. 4i and j ). The enzyme showed highest activity with menadione ( K m = 113.7 μM; V max = 2.0 U, U = nmol/min/mg), and greater activity with short-chain menaquinone ( K m = 139.3 μM; V max = 0.96 U on MK4) than with short-chain ubiquinone ( K m = 7.17 μM; V max = 0.27 U on CoǪ2). These K m (or K 0.5 ) values are comparable to those reported for NDH2 from other organisms, including Saccharomyces cerevisiae (15.2 and 7.9 μM on CoǪ1 and CoǪ2, respectively) 37 and Caldalkalibacillus thermarum (34.0 μM on MD) 38 . C. parvum NDH2 also reduced clofazimine, showing activity comparable to that observed for quinones ( K m = 30.0 μM; V max = 1.0 U). Moreover, clofazimine competed with native substrates, with a relative IC 50 of 2.95 μM for menadione reduction ( Fig. 4k ). Collectively, these biochemical experiments confirmed that C. parvum NDH2 indeed has type 2 NDH activity, and that clofazimine can be reduced by this enzyme. NDH2 localizes to the inner membrane complex and not the mitosome NDH2 typically acts in the respiratory chain and in bacteria is associated with the cell membrane. In eukaryotes including related apicomplexan parasites it is a mitochondrial protein. The C. parvum mitochondrion has lost its genome and much of its respiratory metabolism 39 , this small typically round organelle known as the mitosome is found in close proximity to the nucleus 40 . We were thus surprised to consistently observe NDH2-HA staining as a line close to the surface of parasites, regardless of whether we expressed a transgene or tagged the native locus. High resolution expansion microscopy of infected HCT-8 cultures showed labeling in all intracellular stages of the parasite ( Fig. 5a ). NDH2 labeling outlined extracellular merozoites and male gametes beginning during the intracellular assembly, and in non-dividing parasites appeared as a sharply delineated cap underlying the membrane facing away from the host cell ( Fig. 5a , single arrowhead indicates this cap in a female gamete). Higher magnification reveals this labeling to coincide with the inward facing membrane of the inner membrane complex (IMC, Fig. 5b ) 41 . We confirmed IMC assignment by co-labeling with an antibody to the conserved alveolin domain of IMC proteins 42 , 43 ( Fig. 5c this epitope did not tolerate the expansion protocol). For comparison we also introduced an epitope tag into cgd8_380 which encodes malate oxidoreductase, a presumptive mitosomal protein 44 . For this protein we indeed observed localization to a small organelle close to the nucleus ( Fig. 5d ). We find labeling in all stages with exception of male gametes matching recent findings by Li and colleagues for alternative oxidase 45 . We conclude that NDH2 in C. parvum is not a mitochondrial protein but is localized to the membrane of the IMC facing the parasite cytoplasm. Download figure Open in new tab Figure 5: NDH2 is localized to the inner membrane complex ( a ) Expansion microscopy of HCT-8 cultures infected with NDH2-HA parasites showing protein localization throughout the life cycle. HA in green; NHS-ester in red; Hoechst in blue; scale bar = 10 μm. Single arrowhead NDH2 at the IMC, double arrowhead highlights host-parasite interface at the base of the intracellular parasites in side view. ( b ) Higher magnification detail of the female parasite shown in ( a ) PM = parasite membrane; IMC = inner membrane complex; HA in green; NHS-ester in red; Hoechst in blue; scale bar = 1 μm. Note labeling on the cytoplasmic side of the inner face of the IMC ( c ) Widefield image of NDH2-HA cells labeled antibodies to HA and IMC3 showing colocalization. HA = green; IMC3 = red; scale bar = 5μm. ( d ) Widefield image of MǪA-HA parasites labled with antibodies to HA and IMC3 showing punctate MǪO localization consistent with targeting to the mitosome. HA = green; IMC3 = red; Hoechst = blue; scale bar = 5μm. Genomic heterogeneity at the NDH2 locus and the ΔAA allele are widespread We were initially surprised to find that both parental strains used in the cross carried the ΔAA allele, albeit at different frequencies (30.5% in BG Iowa II and 7.4% in KVI, Fig. 3b and 3c ). This motivated a broader analysis in which we analyzed publicly available whole genomic sequencing (WGS) data from 68 C. parvum and C. hominis sequencing read archives. These were selected for robust genome coverage (>30X mean depth, with mean mapping quality>=60) and broad geographic representation ( Fig. 6a and 6b ). Reads were aligned to the C. parvum reference genome to call variants (INDELs and SNPs with a rigorous quality score >=20 and depth>=20), and we scored the frequency of the ΔAA allele. Remarkably, the same ΔAA variant is detectable in the NDH2 genes of most genomes analyzed (91%) (Supplementary Data 1). In most strains at a frequency around 5-10% while BG Iowa II stands out (note that this genome was sequenced multiple times). We wondered whether this frequency may simply reflect a broader tendency of Cryptosporidium genomes to hypermutation and heterogeneity. As a control, we scored all high impact variations for NDH2 along with three essential and three dispensable genes across 38 C. parvum WGS SRAs of diverse geographic origin. Fig. 6c shows incidence of variation as a heatmap normalized into 10 bins for each gene’s coding sequence region to account for difference in length. The 5’ end of NDH2 clearly stands out, and inspection showed the ΔAA allele to account for all this variation. Download figure Open in new tab Figure 6: NDH2 heterogeneity is widespread and responds dynamically to drug pressure ( a ) Analysis of ΔAA allele frequency across multiple publicly available whole genome sequences of C. parvum and C. hominis strains. Af = Africa; Am = America; Eu = Europe; As = Asia; Oc = Oceania; BG = Bunchgrass Iowa II. Filters were rigorously set to a minimum of 20 unique reads mapped at this location with minimum mapping quality of 60. See Methods section for detail and all data for specific genomes is shown in Supplementary Data 1 along with accession numbers. ( b ) Map showing the global origins of the samples analyzed in ( a ) in orange. ( c ) Heatmap representing high impact variations within the open reading frame of NDH2 and 3 dispensable genes (regular font) and 3 essential genes (italic font). ( d ) Reading frame and amino sequence at the start of NDH2 wildtype (blue) and potential alternative for the ΔAA allele (orange). ( e ) Sanger sequencing and amplicon sequencing of NADH2-HA and NDH2 ΔAA-HA transgenics derived by complementation of the NDH2KO. ( f ) Mean fluorescence of WT BG Iowa II parasites (n = 439) lacking the HA epitope and NHD2-KO parasites expressing NDH2-HA (n = 638) and NDH2 ΔAA-HA (n = 578) in HCT8 cultures labeled with antibody to HA. Statistical analysis was performed using one-way ANOVA and unpaired t test; P value = 0.0004. ( g ) Western blot of WT sporozoites lacking the HA epitope and NHD2-KO sporozoites expressing NDH2-HA or NDH2 ΔAA-HA. Histone 3 served as loading control. Apparent molecular weights of NDH2-HA and NDH2 ΔAA-HA are 72 and 70 kDa, respectively. ( h and i ) IC 50 determination comparing the complemented strains and NDH2 KO ( h ) and the in situ HA-tagged strains homogenizing the NDH2 locus to WT or ΔAA allele ( i ). Data are mean and SD of n=5 biological replicates. ( j ) Amplicon sequencing measuring ΔAA allele frequency across five different batches of wildtype bunchgrass farm oocysts and 33 transgenic oocysts sampled from 2022 to 2025. ( k - m ) Effect of clofazimine treatment on ΔAA allele frequency of BG and KVI strain parasites in tissue culture ( k ) and in infected mice ( l and m ) measured by amplicon sequencing. The ΔAA allele attenuates but does not fully ablate NDH2 activity Analysis of the NDH2 gene shows the ΔAA allele to break the reading frame, however, the use of an alternative start codon might result in a largely intact enzyme with an altered N-terminus ( Fig. 6d ). To better understand the impact of the ΔAA allele we engineered two pairs of parasite strains to differ at this specific position of the gene. These were constructed either by editing the native NDH2 locus to homogenously WT or ΔAA in the BG Iowa II strain, which incurred a small change in the protein sequence as well, or alternatively by complementing the knockout mutant with wildtype NDH2 or NDH2-ΔAA transgenes inserted into a neutral locus (see Supplementary Fig. 2 and Methods for detail). All genotypes were validated by PCR analysis and sequencing ( Fig. 6e ). Engineering these strains, we also introduced HA-epitope tags allowing to measure protein abundance. When imaging infected cultures by immunofluorescence, WT NDH2 is readily observed in all intracellular stages, the ΔAA signal is much lower, however, it is detectable above the level of an untagged control ( Fig. 6f ). Both proteins showed IMC localization. Western blot analysis of protein lysates generated from sporozoites showed a single band of the expected size for WT NDH2-HA and a much weaker, yet again detectable band for NDH2-ΔAA-HA ( Fig. 6g ). The predicted and measured apparent molecular weights of both proteins are near identical. Next, we conducted IC 50 determinations in culture for both strain pairs in direct comparison to the NDH2-KO and found that sole expression of, or complementation with the WT allele produced clofazimine susceptibility with low nanomolar IC 50 s (2.1nM and 6nM, 95% CI=1.4 to 2.9 and 5.7 to 7.2). In contrast, the ΔAA allele conferred resistance in both backgrounds (in situ IC 50 =494nM; 95% CI=292 to 860; ectopic IC 50 =681nM; 95% CI=535 to 885), yet not to the full level of NDH2 deletion ( Fig. 6h and 6i ). We conclude that the ΔAA allele does not result in complete loss of NDH2 activity but severely attenuates its abundance leading to robust resistance. NDH2 allele frequency is dynamic and responds to environmental change We wondered how dynamic the NDH2 locus might be and used amplicon sequencing to measure allele frequencies. We typically purchase BG Iowa II parasites from a commercial vendor and use them to engineer transgenic parasite. The vendor passages BG in immunocompetent calves, while we maintain parasites in ifnγ −/− mice. We measured the ΔAA allele frequency across BG samples from 2022 to 2025 and found a stable mean of 30.2 with a standard deviation of ±2.8, however when analyzing numerous transgenics derived from these parasites over this time frame we measure a very broad distribution ( Fig. 6j ). This suggests that the ΔAA frequency can change, and that the specific ratio might depend on the host environment. To test this further we explored the impact of NDH2 heterogeneity on clofazimine susceptibility. HCT-8 cultures were infected with the BG Iowa II or KVI and grown in the presence or absence of 50 nM clofazimine. Treatment roughly doubled the ΔAA frequency in both strains over 48h ( Fig. 6k ). We also surveyed the allele frequence in infected mice. In the absence of drug treatment, the NDH2 allele frequency remained constant, in contrast, clofazimine treatment resulted in an increase of the ΔAA allele in both strains (reaching 100% for BG Iowa II strain on day 10, Fig. 6l and 6m ). We conclude that NDH2 heterogeneity predisposes to selectable drug resistance, and that the ease and speed with which resistance is achieved may vary among strains, and hinge on the initial level of the ΔAA allele in a population. Discussion Lack of effective treatment remains the crucial challenge to the clinical management of cryptosporidiosis, with consequences most grave for individuals with enhanced susceptibility due to immune deficiency or malnutrition 3 , 6 . The last decade has seen tremendous progress due to the influx of funding and multiple technological advances. Leveraging the resources and expertise of the global malaria drug development effort was highly productive, and many of the strongest leads emerged from screening libraries enriched for compounds that had already shown promise against Plasmodium falciparum 12 , 30 , 46 - 48 . While numerous antimalarials were developed over the last century, malaria parasites have also demonstrated a remarkable ability to evade them, multi-drug resistance is rampant and poses a very serious threat to control 49 . A recent report of Cryptosporidium developing resistance to methionyl t-RNA synthetase inhibition 50 raises concern also for this parasite. Our study suggests that clofazimine, one of the most promising candidates for the treatment of cryptosporidiosis failed due to a preexisting, yet previously undetected drug-resistant allele. We identified differential susceptibility among C. parvum strains, matching previous observations for the single C. hominis strain tested 11 . Forward genetic mapping implicated the NDH2 locus, and biochemical experiments showed the ability of NDH2 to transfer electrons to clofazimine suggesting a prodrug activation mode of action also considered for mycobacteria 19 , 20 . In Mycobacterium NDH2 KO did not yield drug resistance 51 , however, interpretation of this result is complicated by the presence of three NDH type enzymes. In contrast, Cryptosporidium has a single NDH, and we found loss of this enzyme to result in high level resistance, and this loss is readily selectable by drug pressure. Importantly, a resistance conferring allele is already present and globally distributed in the absence of drug pressure. Low bioavailability of clofazimine due to severe diarrhea contributed to its clinical failure 24 – 26 , and additional medicinal chemistry is likely to enhance the formulation and bioavailability of the drug 52 , 53 . However, our animal experimentation shows that depending on the initial allele frequency, resistance can be attained over the course of a single infection, which casts serious doubt on the prospect of clofazimine as an anti- Cryptosporidium agent. Overall, this suggests that considering the resistance potential is an important step in future preclinical evaluation of anti- Cryptosporidium drugs. NADH including type 2 NDH typically function as part of the respiratory chain transferring electrons to a lipophilic quinone acceptor in the bacterial plasma membrane, or in eukaryotes, the inner mitochondrial membrane. In mycobacteria the enzyme is dispensable, if fatty acids are not a main carbon source 51 . In apicomplexan parasites NADH2 replaces the canonical complex I found in many other eukaryotes 44 , 54 . T. gondii has two NDH2 enzymes that are both dispensable 55 , the single Plasmodium NDH2 can be ablated with little consequence to blood stages, however development in the mosquito is blocked 56 , 57 . In other apicomplexans NDH2 is a mitochondrial enzyme, while in Cryptosporidium it is localized to the IMC underlying the parasites plasma membrane. Interestingly, it appears confined to one face of the IMC. This was an initially surprising finding but matches the recent spatial proteomic assignment 58 . In contrast, alternative oxidase and malate oxidoreductase, the two other redox enzymes thought to use a quinol electron carrier in Cryptosporidium are indeed mitochondrial proteins ( 45 and Fig. 5d ). The C. parvum mitochondrion is highly reduced, it has lost its genome, the TCA cycle and most of the electron transport chain, and even those elements remaining appear dispensable 45 . The function of NDH2 at the IMC membrane is unknown, but its relocation out of the mitosome likely deemphasizes its importance to mitochondrial respiration. In the facultative intracellular pathogen Listeria monocytogenes NDH2 impacts redox balance and virulence independent of the respiratory chain to adjust to different host niches and metabolic environments 59 . It is thus possible that NDH2 and ubiquinone might mitigate oxidative stress in a changing metabolic environment, and its main role may be to balance the cytoplasmic NAD/NADH pool. The changed localization of NDH2 could reflect a more outward facing role in attaining and modifying critical metabolites. Multiple recent studies have found Cryptosporidium to interact with host and microbiome derived metabolites in ways that profoundly impact parasite survival and growth 60 – 63 . NDH2 could play a role in detoxifying detrimental metabolites 64 . Lastly, redox pathways play key roles in antimicrobial restriction and broader immune signaling, and NDH2 activity may interact with and modulate these pathways 65 . Why is genomic heterogeneity at the NDH2 locus and the specific ΔAA allele conserved and widespread? We propose that heterogeneity may represent a metabolic rheostat and might carry the benefit of adaptability. When BG Iowa II parasites are passaged in calves, they carried a high frequence of the attenuated ΔAA allele and this remained constant over years. Upon introduction into mice this frequency varied significantly. What exactly drives this change is not known yet, but host metabolism, host nutrition (high fat milk replacer versus rodent chow), divergent microbiome composition, or differential immune pressure could all impact on parasite redox balance. Apicomplexa are widely seen as transcriptionally hard-wired with only limited metabolic flexibility 66 , 67 . Cryptosporidium is unique among apicomplexans in that it undergoes obligate sex every two days resulting in a very high rates of rapid recombination 68 , 69 . Combining this feature with genomic heterogeneity may allow the population in a single host to act as functional diploid with the ability to dial up or down a particular allele to dynamically adjust to change. Online Methods Parasites C. parvum isolates and derived transgenics used in this study were obtained and genotyped as described 70 . Original sources are: BG Iowa II, Bunch Grass Farms, Deary, Idaho; ST Iowa II, Dr. Reed, University of Arizona; INRA, Dr. Fabrice Laurent, INRAE and University of Tours, Nouzilly, France; KVI, Dr. Yasur-Landau, Beit Dagab, Israel. Generation of transgenic strains Guide oligo nucleotides (Sigma-Aldrich) were introduced into the C. parvum Cas9/U6 plasmid 28 by restriction cloning (see reference 71 for guide design) and repair templates were constructed by Gibson assembly (New England Biolabs). Excysted sporozoites were transfected as described in 71 . Oligos used for genotyping can be found in Supplementary Data 3. Ablation of NDH2 The insert encodes Nluc followed by the neomycin phosphotransferase drug-selection marker and was knocked into the NDH2 locus to induce a knockout. Guide: 7_1900_guide_F / 7_1900_guide_R Repair template: NDH2KO_F / NDH2KO_R NDH2 as a selection marker The insert encodes Nluc followed by a tdNeonGreen. Guide: 7_1900_guide_F / 7_1900_guide_R Repair template: mNG_Cfz_F / mNG_Cfz_R NDH2 in situ HA-epitope The insert encodes a recodonized version of NDH2 (position 81 to stop codon) with a triple HA-epitope sequence followed by Nluc and the neomycin phosphotransferase drug-selection marker. 5’ homology arms between the NDH2-HA and the NDH2-ΔAA-HA strains differ. Note that the NDH2-ΔAA-HA strain has a slightly altered amino sequence at the beginning of the transgene ( Fig. 6d ). NDH2-HA guide: 7_1900_guide_F / 7_1900_guide_R NDH2-ΔAA-HA guide: NDH2_guide_2_F / NDH2_guide_2_R NDH2-HA repair template: 7_1900_repair_+AA_F / 7_1900_repair_R NDH2-ΔAA-HA repair template: NDH2_-AA_2_F / 7_1900_repair_R Ectopic expression of NDH2-HA The inserts encode the last 113 bp of the pheRS gene (cgd3_3320, recodonized) including the mutation that confers resistance (L482V) to BRD7929. This short sequence is followed by the whole NDH2 gene cassette (including its own promoter, and a triple HA-epitope sequence and the enolase 3’UTR). The only difference between the repairs is the presence or absence of two adenines at position 81 ( Supplementary Fig. 2b ) Guide: PheSF_guide_New_SV / PheSR_guide_New_SV Repair template: lift_NDH2_aldo3utr_F_new / lift_NDH2_3xHA_R Cgd8_380 in situ HA-epitope The insert encodes for a triple HA epitope followed by Nluc and the neomycin phosphotransferase drug-selection marker. We also included a reverse COWP1 3’UTR at the 3’ end of the repair to assure correct expression of the next downstream gene which is transcribed on the minus strand ( Supplementary Fig. 2b ). Guide: 8_380_guide_F/8_380_guide_R Repair template: 8_380_tag_F_NEW/8_380_tag_R_NEW Cell culture and Cryptosporidium infections HCT-8 cells were purchased from ATCC (CCL-224TM) and maintained in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% Cosmic calf serum (HyClone) at 37°C in presence of 5% CO 2 . Oocysts were treated with 10 mM HCl at 37°C for 45-60 minutes before washing and resuspension in medium containing 1% serum, 0.2 mM sodium taurocholate, and 20mM sodium bicarbonate (infection media) to induce excystation. Infection media containing oocysts were transferred immediately onto cells and remained for the duration of the infection. Dose-response assay and IC50 calculations In 96-well plates, HCT-8 cells were infected with 10,000 oocysts per well and incubated at 37°C for 3 hours. Equivalent volumes of clofazimine (2X final concentration) or 0.5% DMSO in infection media were added to the wells and incubated at 37°C for 48 hours. Medium was aspirated, cells were lysed and mixed with NanoGlo substrate (Promega), and luminescence was measured using a Glomax reader (Promega) (REF). IC50 values were calculated in GraphPad Prism software v9 (at least two independent experiments, each conducted with 5 replicates). Mouse infections and clofazimine treatment All mouse infections were performed using 4- to 8-week-old male and female Ifnγ −/− (Jackson Laboratory stock no. 002287) mice bred in-house. Mice were pretreated with antibiotic water and infected via oral gavage as detailed in 28 , 71 . Clofazimine (Sigma-Aldrich cat no. C8895) was formulated in MC-Tween (0.5% methylcellulose and 0.5% Tween-80) or PEG-Glucose (70% polyethylene glycol 400 and 1.5% glucose) suspension and given to mice orally. Feces of clofazimine- and vehicle-treated mice were collected and pooled as shown in Fig. 1F, 1G . Oocyst purification and genomic DNA extraction Fecal material of infected mice was collected and oocyst were purified using a sucrose gradient and CsCl flotation 71 . Genomic DNA was extracted using phenol/chloroform as described 29 . Library preparation and Illumina sequencing of genomic DNA from cross progeny We prepared Illumina libraries from extracted genomic DNA and sequenced both parents and 6 segregant pools. The library preparation was carried out using Illumina DNA Prep (former Nextera DNA Flex kit, Illumina Inc.). Subsequently, sequencing was performed on the Illumina NextSeq 2000 sequencer, utilizing P2 300 cycle flowcell kit. Genotype calling The recent Iowa II telomere to telomere de novo assembly 72 was used as reference genome to identify single-nucleotide polymorphism (SNP) distinguishing the two parents, which were then used for bulk segregant analysis. Whole-genome sequencing reads for each library were individually mapped to the Iowa II de novo assembly using the BWA-MEM alignment algorithm with default parameters 73 . The resulting alignments were converted to SAM format, sorted into BAM format, and deduplicated using Picard tools 8 . Variants for each sample were called using HaplotypeCaller from GATK Suite and were subsequently aggregated across all samples using GenotypeGVCFs 74 (see https://github.com/ruicatxiao/Automated_Bulk_Segregant_Analysis for detailed parameters). Bulk segregant analysis SNP loci with coverage below 30× in either of the compared pools were excluded from bulk segregant analysis. At each variable locus, we counted reads corresponding to the genotypes of each parent and calculated allele frequencies. Iowa II allele frequencies were plotted across the genome, and outliers were removed using Hampel’s rule with a window size of 100 loci. Bulk segregant analysis was performed using the R package ǪTLseqr 75 . Extreme ǪTLs were defined as loci with false discovery rates (FDRs, Benjamini-Hochberg adjusted p -values) below 0.01. Summary of the analysis and all metadata can be found in Supplementary Data 2. cgd7_1G00 ΔAA allele frequency analysis using publicly available sequence read archives We have developed a Python pipeline, sra2vcf ( https://github.com/ruicatxiao/sra2vcf ), that performs robust and comprehensive SNP/INDEL analysis on both local and online SRA datasets for long-read or short-read DNA/RNA sequencing. Briefly for each sample the pipeline use sra-toolkit to download SRA and BWA is used to map reads to the genome. Mapped reads are sorted by genome coordinates using SAMTools, then duplicated reads are marked and removed with GATK suite. Bcftools mpileup is used to call variants using an INDEL detection optimized illumina-1.20 model. The output vcf are filtered by variant coverage and quality. Individual SRA sample’s vcf are aggregated to generate the final output table. The pipeline can accumulate and add new samples without reprocessing existing ones, and it intelligently checks for existing intermediate outputs to avoid redundant computations. High impact variant analysis for essential and non-essential genes using publicly available SRAs The sra2vcf pipeline generates individual VCF files that are first processed with SnpEff to annotate variant effects on protein coding frames for each sample, followed by SnpSift aggregation to identify HIGH impact variants shared across samples. We developed Python programs goi_af.py and goi_cov.py to analyze SnpEff-annotated VCF files along with a gene-of-interest list. The output reports allele frequencies and allele coverage for each HIGH impact variant. We divide each gene’s coding regions into 10 bins from 5’ to 3’ to generate comprehensive allele frequency tables for target genes of varying length. All analysis codes are publicly available at https://github.com/ruicatxiao/cparvum_ndh2_clofazimine-resistance . Nanopore sequencing of amplified genomic material from cell cultures and fecal samples Parasite genomic DNA was extracted from either cell culture supernatants or fecal samples as described in 29 . PCR amplification of the NDH2 locus was performed using PrimeStar Max ver. 2 (fwd primer: 5’-TCAAGTGGGGTCTCGGATG-3’; rev primer: 5’-CCCCACCCAGTACCTAAGATG-3’), then purified using Bioneer AccuPrep Gel/PCR Purification kit before sequencing. Nanopore sequencing of purified PCR amplicons was conducted using a commercial service provided by Eurofins Genomics. Genomic sequencing analyses Raw read fastq files were mapped to the BG Iowa II de novo assembly using the BWA-MEM alignment algorithm with default parameters 73 . The resulting alignments were converted to SAM format, sorted into BAM format, and deduplicated using Picard tools 8 . Alignments were visualized in IGV to calculate deletion frequencies. All codes used for this analysis are available through github repository https://github.com/ruicatxiao/cparvum_ndh2_clofazimine-resistance . Engineering transgenic strains Guide oligonucleotides (Sigma-Aldrich) were introduced into the C. parvum Cas9/U6 plasmid by restriction cloning and repair templates were constructed by Gibson assembly (New England Biolabs) 71 and excysted sporozoites were transfected as described 71 . Briefly, 1.56 × 10 7 Cp BG Iowa II oocysts or 5 x 10 6 Cp KVI oocysts were incubated at 37 °C for 1 hr in 10 mM HCl followed by two washes with phosphate buffered saline (PBS) and an incubation at 37 °C for 1 h in 0.2 mM sodium taurocholate and 20 mM sodium bicarbonate to induce excystation 29 . Excysted sporozoites were electroporated and used to infect mice as described 29 . Integration was validated by PCR mapping and/or Sanger sequencing. Immunofluorescence assay HCT-8 cells were seeded on coverslips in 24-well plates prior to infection. Infected coverslips were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) in PBS for 20 min and then permeabilized with 0.25% Triton X in PBS for 10 min at room temperature. Coverslips were then blocked with 1% bovine serum albumin (BSA) in PBS for 1 hr before primary antibody (1:1000 rat anti-HA,1:500 rabbit anti-IMC3 or 1:1000 biotinylated anti-VVL) incubation followed by secondary antibody ( 1:1000 Alexa Fluor 488 anti-rat, 1:1000 Alexa Fluor 594 anti-rabbit or 1:1000 Alexa Fluor 594 streptavidin) incubation, both for 1 hr in 1% BSA. Coverslips were mounted using fluoro-gel mounting medium (Electron Microscopy Sciences) and imaged using the widefield Leica DM6000B or GE Delta Vision OMX. Ultrastructure expansion microscopy Ultrastructure expansion microscopy was applied to Cryptosporidium infected HCT-8 cells as described for sporozoites 58 . Infected coverslips were fixed in 4% paraformaldehyde for 20 min at 25°C and washed thrice with PBS before incubating overnight at 37°C in acrylamide and formaldehyde to prevent protein crosslinking. Samples were embedded in a water-based gel then denatured at 95°C prior to expansion in water to 4-5X their original size. Gels were shrunk in PBS before blocking and staining to save reagents. After re-expansion in water, the gels were imaged using the Leica Stellaris FALCON confocal microscope. Biochemical assays Recombinant CpNDH2 was expressed as a maltose-binding protein (MBP) fusion using the pMAL-c5X vector (New England Biolabs, Ipswich, MA, USA). Clofazimine (purity ≥99%) and NADH (purity ≥98%) were purchased from Yuanye Bio-Technology (Shanghai, China). Menadione (purity ≥99.5%) was obtained from MedChemExpress (New Jersey, USA). Ubiquinone-2 (CoǪ2; purity ≥95%) and menaquinone-4 (MK4; purity ≥98%) were purchased from GlpBio (Shanghai, China). For cloning of the CpNDH2 gene (gene ID: cgd7_1900), the full-length open reading frame encoding wild-type CpNDH2 (CpNDH2-WT) was amplified from C. parvum genomic DNA ( GpC0 subtype IIdA19G1) by PCR using Phanta Max Super-Fidelity DNA Polymerase (Vazyme International, Nanjing, Jiangsu, China). The primers used were CpNDH2-forward (5′-cgcgatatcgtcgacggatccATGTCTAACTCTGAAAAGAATACTTCCAA-3′) and CpNDH2-reverse (5′-agcttatttaattacctgcagTTAGTGAGAAACGTTCATTTTGTAGATT-3′). Lowercase letters indicate additional sequences included for seamless cloning. The PCR amplicon was assembled into pMAL-c5X using the LightNing DNA Assembly Mix Plus kit (BestEnzymes Biotech, Lianyungang, Jiangsu, China). Recombinant plasmids were propagated in E. coli TOP10, purified, and sequence-verified for correct insertion. For protein expression, the verified construct was transformed into E. coli BL21(DE3). Cultures were grown at 37 °C to an OD 450 of ∼0.6, then induced with 0.5 mM IPTG at 25 °C for 6 h. Cells were harvested, lysed by sonication, and recombinant proteins purified using amylose-resin columns according to the manufacturer’s instructions (New England Biolabs). The quality and yield of purified proteins were assessed by SDS-PAGE and Bradford assay, using bovine serum albumin as the standard. Tag-free CpNDH2 was prepared by cleavage of MBP-CpNDH2 with factor Xa protease (New England Biolabs). Reactions were carried out at room temperature for 24 h in 200 μL of buffer containing 20 mM Tris (pH 8.0), 100 mM NaCl, 2 mM CaCl₂, 200 μg MBP-CpNDH2, and 4 μg factor Xa. Following cleavage, proteins were dialyzed against buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA) at 4 °C for 12 h, and the MBP tag was removed by amylose-resin purification. MBP alone was similarly expressed and purified for use as a negative control and for background subtraction in assays. The catalytic activity of CpNDH2-WT was measured using a spectrophotometric assay adapted from published protocols 34 – 36 . Reactions were carried out at 25 °C in 100 μL of buffer containing 50 mM Tris (pH 7.0), 1 mM EDTA, 0.1% Triton X-100, 400 μM NADH, 4 μM CpNDH2 (MBP-fusion or tagless), and substrate at the indicated concentrations. Reactions were initiated by substrate addition, and NADH oxidation was monitored at OD 340 in a microplate reader (BioTek Instruments, Santa Clara, CA, USA) at 0.5-min intervals for up to 40 min. Since MBP-tagged and tag-free CpNDH2 displayed comparable activity toward menadione (MD) ( Supplemental Fig. 1 ), subsequent assays with MK4, CoǪ2, and clofazimine (CFZ) were performed using intact MBP-CpNDH2-WT. Optical density data were plotted against substrate concentrations, and kinetic parameters were estimated by nonlinear regression using a sigmoidal curve fit that incorporated the Hill coefficient. This yielded K 0.5 (or K ′) and V max values. When the Hill slope was close to 1.0, the reaction followed Michaelis–Menten kinetics and K 0.5 was equivalent to K m . Western blotting Western blot was performed using rabbit anti-HA antibody diluted 1/1000 and mouse anti-H3pan antibody diluted 1/1000 and secondary anti-rabbit IRDye 800 and anti-mouse IRDye 680 diluted 1/10000. We followed the Licor best practice protocol. Blots were imaged using an Odyssey Licor device. Flow cytometry 1 million purified oocysts were washed and resuspended in 200 μL FACS buffer (1× PBS, 0.2% bovine serum albumin, 1 mM EDTA). Oocysts were then passed through a 70 μM mesh filter. Data were collected on a FACSymphony A3 Lite (BD Biosciences) and analyzed with FlowJo v10 software (TreeStar). Construct integration frequency was measured by positivity for an mNeonGreen reporter. Contributions Conceptualization: B.S., G.Y.B., S.S. Methodology: A.B., K.M.O., P.J., B.X., D.W., R.X., G.Y.B., S.S. Investigation: A.B. (expansion microscopy, Fig. 5a–b ); K.M.O. (flow cytometry, Fig. 4g ); P.J., B.X., D.W. (biochemical assays, Fig. 4i–k and Supplementary Fig. 1 ); R.X. (allele frequency calculations, Fig. 6a–c ); G.Y.B., S.S. (all other experiments). Formal analysis: R.X., G.Y.B., S.S., A.B., K.M.O., P.J., B.X., D.W. Visualization: G.Y.B., S.S., B.S. Writing original draft: G.Y.B., S.S., B.S. Writing review C editing: All authors. Supervision: B.S. Data availability Whole genome raw sequencing data and the raw amplicon sequencing data have been deposited in the NCBI’s Sequencing Read Archive database under Bioproject numbers PRJNA1336748 and PRJNA1337473, respectively. Supplementary Information Download figure Open in new tab Supplementary Figure 1: Initial assessment of catalytic activity using purified recombinant wild-type CpNDH2 protein in intact and cleaved forms (a) SDS-PAGE gels showing purified intact (uncleaved) and cleaved MBP-CpNDH2 protein. (b) Initial assessment of intact and cleaved CpNDH2 proteins in catalyzing the electron transfer from NADH to menadione (MD) and clofazimine (CFZ) using a spectrometeric assay. (c) Chemical structures of menadione, menaquinone, ubiquinone, and clofazimine. Green stars indicate atoms positioned to accept electrons during enzymatic reduction. Download figure Open in new tab Supplementary Figure 2: Transgenic parasite strains constructed for this study ( a ) Map of NDH2 insitu HA epitope tagging strategy. Two strains were generated, either with or without INDEL (plus or minus AA). HA epitope = green. ( b ) Gel shows PCR mapping of the plus AA strain described in (a). ( c ) Gel shows PCR mapping of the minus AA strain described in (a). ( d ) Ectopic expression of NDH2-HA and NDH2-ΔAA-HA. HA epitope = green. ( e ) Gel shows PCR mapping of the strains described in (d). (d). ( f ) Map of MǪO insitu HA epitope tagging strategy. HA epitope = green. ( g ) Gel shows PCR mapping of the MQO insitu HA epitope strain. Labelling of the gel refers to the amplicons shown in the maps (f). Labelling of all the gels refer to the amplicons shown in the respective maps. Acknowledgements This work was supported in part by grants from the National Institutes of Health to B.S. (R01AI112427, R01AI127798), B.S. and Christopher Hunter (R01AI148249), to the Penn Vet Imaging Core (S10OD021633), Swiss National Science Foundation fellowships 402 P2BEP3_191774 and P500PB_211097 to S.S, and European Molecular Biology Organization fellowship ALTF1145-2021 to A.B. We thank Adam Sateriale for initial contributions, Jessica Byerly and Chloe Tang for support of animal experimentation, and Bethan Wallbank, Allison Cohen, Abigail Daniels, Chloe Tang, and Maria Merolle for sharing transgenic parasites. Funder Information Declared National Institutes of Health , R01AI112427 , R01AI127798 , R01AI148249 Swiss National Science Foundation, https://ror.org/00yjd3n13 , P2BEP3_191774 , P500PB_211097 European Molecular Biology Organization, https://ror.org/04wfr2810 , ALTF1145-2021 References 1. ↵ Centers for Disease, C . Cryptosporidiosis: assessment of chemotherapy of males with acquired immune deficiency syndrome (AIDS) . MMWR Morb Mortal Wkly Rep 31 , 589 – 592 ( 1982 ). OpenUrl PubMed 2. ↵ Cohn , I. S. , Henrickson , S. E. , Striepen , B. C Hunter , C. A. Immunity to Cryptosporidium: Lessons from Acquired and Primary Immunodeficiencies . J Immunol 20G , 2261 – 2268 ( 2022 ). doi: 10.4049/jimmunol.2200512 OpenUrl CrossRef 3. ↵ Checkley , W. et al. A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for cryptosporidium . Lancet Infect Dis 15 , 85 – 94 ( 2015 ). doi: 10.1016/S1473-3099(14)70772-8 OpenUrl CrossRef PubMed 4. ↵ Kotloff , K. L. et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study . Lancet 382 , 209 – 222 ( 2013 ). doi: 10.1016/S0140-6736(13)60844-2 OpenUrl CrossRef PubMed Web of Science 5. ↵ Khalil , I. A. et al. Morbidity, mortality, and long-term consequences associated with diarrhoea from Cryptosporidium infection in children younger than 5 years: a meta-analyses study . Lancet Glob Health 6 , e758 – e768 ( 2018 ). doi: 10.1016/S2214-109X(18)30283-3 OpenUrl CrossRef PubMed 6. ↵ Gilbert , I. H. et al. Safe and effective treatments are needed for cryptosporidiosis, a truly neglected tropical disease . BMJ Glob Health 8 ( 2023 ). doi: 10.1136/bmjgh-2023-012540 OpenUrl FREE Full Text 7. Huston , C. D. et al. A Proposed Target Product Profile and Developmental Cascade for New Cryptosporidiosis Treatments . PLoS neglected tropical diseases G , e0003987 ( 2015 ). doi: 10.1371/journal.pntd.0003987 OpenUrl CrossRef PubMed 8. ↵ Ashigbie , P. G. et al. Use-case scenarios for an anti-therapeutic . PLoS neglected tropical diseases 15 ( 2021 ). ARTN e0009057 doi: 10.1371/journal.pntd.0009057 OpenUrl CrossRef 9. ↵ Love , M. S. C Choy , R. K. M. Emerging treatment options for cryptosporidiosis . Current opinion in infectious diseases ( 2021 ). doi: 10.1097/ǪCO.0000000000000761 OpenUrl CrossRef 10. ↵ Bessoff , K. , Sateriale , A. , Lee , K. K. C Huston , C. D. Drug repurposing screen reveals FDA-approved inhibitors of human HMG-CoA reductase and isoprenoid synthesis that block Cryptosporidium parvum growth . Antimicrob Agents Chemother 57 , 1804 – 1814 ( 2013 ). doi: 10.1128/AAC.02460-12 OpenUrl Abstract / FREE Full Text 11. ↵ Love , M. S. et al. A high-throughput phenotypic screen identifies clofazimine as a potential treatment for cryptosporidiosis . PLoS neglected tropical diseases 11 , e0005373 ( 2017 ). doi: 10.1371/journal.pntd.0005373 OpenUrl CrossRef PubMed 12. ↵ Manjunatha , U. H. et al. A Cryptosporidium PI(4)K inhibitor is a drug candidate for cryptosporidiosis . Nature 546 , 376 – 380 ( 2017 ). doi: 10.1038/nature22337 OpenUrl CrossRef PubMed 13. ↵ Xu , J. , Koval , A. C Katanaev , V. L. Clofazimine: A journey of a drug . Biomed Pharmacother 167 , 115539 ( 2023 ). doi: 10.1016/j.biopha.2023.115539 OpenUrl CrossRef PubMed 14. ↵ Chemotherapy of leprosy for control programmes. World Health Organ Tech Rep Ser 675 , 1 - 33 ( 1982 ). OpenUrl PubMed 15. ↵ Guglielmetti , L. et al. Bedaquiline, delamanid, linezolid, and clofazimine for rifampicin-resistant and fluoroquinolone-resistant tuberculosis (endTB-Ǫ): an open-label, multicentre, stratified, non-inferiority, randomised, controlled, phase 3 trial . Lancet Respir Med ( 2025 ). doi: 10.1016/S2213-2600(25)00194-8 OpenUrl CrossRef 16. ↵ World Health Organization: Key updates to the treatment of drug-resistant tuberculosis: rapid communication , June 2024 . ( World Health Organization , 2024). 17. ↵ Stadler , J. A. M. , Maartens , G. , Meintjes , G. C Wasserman , S. Clofazimine for the treatment of tuberculosis . Front Pharmacol 14 , 1100488 ( 2023 ). doi: 10.3389/fphar.2023.1100488 OpenUrl CrossRef 18. ↵ Morrison , N. E. C Marley , G. M. The mode of action of clofazimine DNA binding studies . Int J Lepr Other Mycobact Dis 44 , 133 – 134 ( 1976 ). OpenUrl PubMed 19. ↵ Yano , T. et al. Reduction of clofazimine by mycobacterial type 2 NADH:quinone oxidoreductase: a pathway for the generation of bactericidal levels of reactive oxygen species . J Biol Chem 286 , 10276 – 10287 ( 2011 ). doi: 10.1074/jbc.M110.200501 OpenUrl Abstract / FREE Full Text 20. ↵ Lechartier , B. C Cole , S. T. Mode of Action of Clofazimine and Combination Therapy with Benzothiazinones against Mycobacterium tuberculosis . Antimicrob Agents Chemother 5G , 4457 – 4463 ( 2015 ). doi: 10.1128/AAC.00395-15 OpenUrl Abstract / FREE Full Text 21. ↵ Van Rensburg , C. E. , Joone , G. K. , O’Sullivan , J. F. C Anderson , R. Antimicrobial activities of clofazimine and B669 are mediated by lysophospholipids . Antimicrob Agents Chemother 36 , 2729 – 2735 ( 1992 ). doi: 10.1128/AAC.36.12.2729 OpenUrl Abstract / FREE Full Text 22. Steel , H. C. , Matlola , N. M. C Anderson , R. Inhibition of potassium transport and growth of mycobacteria exposed to clofazimine and B669 is associated with a calcium-independent increase in microbial phospholipase A2 activity . J Antimicrob Chemother 44 , 209 – 216 ( 1999 ). doi: 10.1093/jac/44.2.209 OpenUrl CrossRef PubMed Web of Science 23. ↵ De Bruyn , E. E. , Steel , H. C. , Van Rensburg , E. J. C Anderson , R. The riminophenazines, clofazimine and B669, inhibit potassium transport in gram-positive bacteria by a lysophospholipid-dependent mechanism . J Antimicrob Chemother 38 , 349 – 362 ( 1996 ). doi: 10.1093/jac/38.3.349 OpenUrl CrossRef PubMed Web of Science 24. ↵ Iroh Tam , P. , et al. Clofazimine for Treatment of Cryptosporidiosis in Human Immunodeficiency Virus Infected Adults: An Experimental Medicine, Randomized, Double-blind, Placebo-controlled Phase 2a Trial . Clin Infect Dis 73 , 183 – 191 ( 2021 ). doi: 10.1093/cid/ciaa421 OpenUrl CrossRef PubMed 25. ↵ Zhang , C. X. et al. Clofazimine pharmacokinetics in HIV-infected adults with diarrhea: Implications of diarrheal disease on absorption of orally administered therapeutics . CPT Pharmacometrics Syst Pharmacol 13 , 410 – 423 ( 2024 ). doi: 10.1002/psp4.13092 OpenUrl CrossRef PubMed 26. ↵ Zhang , C. X. et al. Pharmacokinetics and Pharmacodynamics of Clofazimine for Treatment of Cryptosporidiosis . Antimicrob Agents Chemother 66 , e0156021 ( 2022 ). doi: 10.1128/AAC.01560-21 OpenUrl CrossRef PubMed 27. ↵ Shaw , S. et al. Genetic crosses reveal genomic loci responsible for virulence in Cryptosporidium parvum infection Cell Rep 44 , 116315 ( 2025 ). doi: 10.1016/j.celrep.2025.116315 OpenUrl CrossRef PubMed 28. ↵ Vinayak , S. et al. Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum . Nature 523 , 477 – 480 ( 2015 ). doi: 10.1038/nature14651 OpenUrl CrossRef PubMed 29. ↵ Shaw , S. et al. Genetic crosses within and between species of Cryptosporidium . Proc Natl Acad Sci U S A 121 , e2313210120 ( 2024 ). doi: 10.1073/pnas.2313210120 OpenUrl CrossRef PubMed 30. ↵ Vinayak , S. et al. Bicyclic azetidines kill the diarrheal pathogen Cryptosporidium in mice by inhibiting parasite phenylalanyl-tRNA synthetase . Sci Transl Med 12 ( 2020 ). doi: 10.1126/scitranslmed.aba8412 OpenUrl Abstract / FREE Full Text 31. ↵ Baptista , R. P. , Xiao , R. , Li , Y. , Glenn , T. C. C Kissinger , J. C. New T2T assembly of Cryptosporidium parvum IOWA II annotated with Legacy-Compatible Gene identifiers . Sci Data 12 , 1039 ( 2025 ). doi: 10.1038/s41597-025-05364-3 OpenUrl CrossRef PubMed 32. ↵ Li , X. , Kumar , S. , Brenneman , K. V. C Anderson , T. J. C. Bulk segregant linkage mapping for rodent and human malaria parasites . Parasitology international G1 , 102653 ( 2022 ). doi: 10.1016/j.parint.2022.102653 OpenUrl CrossRef PubMed 33. ↵ Cen , Y. et al. Novel roles of RNA-binding proteins in drug resistance of breast cancer: from molecular biology to targeting therapeutics . Cell Death Discov G , 52 ( 2023 ). doi: 10.1038/s41420-023-01352-x OpenUrl CrossRef 34. ↵ Murugesan , D. et al. 2-Mercapto-Ǫuinazolinones as Inhibitors of Type II NADH Dehydrogenase and Mycobacterium tuberculosis: Structure-Activity Relationships, Mechanism of Action and Absorption, Distribution, Metabolism, and Excretion Characterization . ACS Infect Dis 4 , 954 – 969 ( 2018 ). doi: 10.1021/acsinfecdis.7b00275 OpenUrl CrossRef PubMed 35. Nies , S. C. et al. Systems Analysis of NADH Dehydrogenase Mutants Reveals Flexibility and Limits of Pseudomonas taiwanensis VLB120’s Metabolism . Appl Environ Microbiol 86 ( 2020 ). doi: 10.1128/AEM.03038-19 OpenUrl Abstract / FREE Full Text 36. ↵ Shirude , P. S. et al. Ǫuinolinyl Pyrimidines: Potent Inhibitors of NDH-2 as a Novel Class of Anti-TB Agents . ACS medicinal chemistry letters 3 , 736 – 740 ( 2012 ). doi: 10.1021/ml300134b OpenUrl CrossRef PubMed 37. ↵ Murai , M. et al. Characterization of the ubiquinone binding site in the alternative NADH-quinone oxidoreductase of Saccharomyces cerevisiae by photoaffinity labeling . Biochemistry 4G , 2973 – 2980 ( 2010 ). doi: 10.1021/bi100005j OpenUrl CrossRef 38. ↵ Blaza , J. N. et al. The mechanism of catalysis by type-II NADH:quinone oxidoreductases . Scientific reports 7 , 40165 ( 2017 ). doi: 10.1038/srep40165 OpenUrl CrossRef PubMed 39. ↵ Liu , S. et al. Evolution of mitosome metabolism and invasion-related proteins in Cryptosporidium . BMC Genomics 17 , 1006 ( 2016 ). doi: 10.1186/s12864-016-3343-5 OpenUrl CrossRef PubMed 40. ↵ Riordan , C. E. , Ault , J. G. , Langreth , S. G. C Keithly , J. S. Cryptosporidium parvum Cpn60 targets a relict organelle . Curr Genet 44 , 138 – 147 ( 2003 ). OpenUrl CrossRef PubMed Web of Science 41. ↵ Ostrovska , K. C Paperna , I. Cryptosporidium sp. of the starred lizard Agame stellio:ultrastructure and life cycle . Parasitology Research 76 , 712 – 720 ( 1990 ). OpenUrl CrossRef PubMed 42. ↵ Gubbels , M. J. , Wieffer , M. C Striepen , B. Fluorescent protein tagging in Toxoplasma gondii: identification of a novel inner membrane complex component conserved among Apicomplexa . Mol Biochem Parasitol 137 , 99 – 110 ( 2004 ). doi: 10.1016/j.molbiopara.2004.05.007S0166685104001471 [pii] OpenUrl CrossRef PubMed Web of Science 43. ↵ Gould , S. B. , Tham , W. H. , Cowman , A. F. , McFadden , G. I. C Waller , R. F. Alveolins, a new family of cortical proteins that define the protist infrakingdom Alveolata . Mol Biol Evol 25 , 1219 – 1230 ( 2008 ). msn070 [pii] doi: 10.1093/molbev/msn070 OpenUrl CrossRef PubMed Web of Science 44. ↵ Mogi , T. C Kita , K. Diversity in mitochondrial metabolic pathways in parasitic protists Plasmodium and Cryptosporidium . Parasitology international 5G , 305 – 312 ( 2010 ). doi: 10.1016/j.parint.2010.04.005 OpenUrl CrossRef 45. ↵ Li , N. et al. Biological Characterization of Alternative Oxidase in Cryptosporidium Mitosome . FASEB J 3G , e71000 ( 2025 ). doi: 10.1096/fj.202501254R OpenUrl CrossRef 46. ↵ Baragana , B. et al. Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis . P Natl Acad Sci USA 116 , 7015 – 7020 ( 2019 ). doi: 10.1073/pnas.1814685116 OpenUrl Abstract / FREE Full Text 47. Lee , S. et al. Piperazine-Derivative MMV665917: an effective drug in the diarrheic piglet model of Cryptosporidium hominis . J Infect Dis ( 2019 ). doi: 10.1093/infdis/jiz105 OpenUrl CrossRef PubMed 48. ↵ Jumani , R. S. et al. A Novel Piperazine-Based Drug Lead for Cryptosporidiosis from the Medicines for Malaria Venture Open-Access Malaria Box . Antimicrob Agents Chemother 62 ( 2018 ). doi: 10.1128/AAC.01505-17 OpenUrl Abstract / FREE Full Text 49. ↵ Rosenthal , P. J. et al. The emergence of artemisinin partial resistance in Africa: how do we respond? Lancet Infect Dis 24 , e591 – e600 ( 2024 ). doi: 10.1016/S1473-3099(24)00141-5 OpenUrl CrossRef PubMed 50. ↵ Hasan , M. M. et al. Spontaneous Selection of Cryptosporidium Drug Resistance in a Calf Model of Infection . Antimicrob Agents Chemother 65 ( 2021 ). doi: 10.1128/AAC.00023-21 OpenUrl Abstract / FREE Full Text 51. ↵ Beites , T. et al. Plasticity of the Mycobacterium tuberculosis respiratory chain and its impact on tuberculosis drug development . Nature communications 10 , 4970 ( 2019 ). doi: 10.1038/s41467-019-12956-2 OpenUrl CrossRef PubMed 52. ↵ Kandagatla , H. P. et al. Highly increasing solubility of clofazimine, an extremely water-insoluble basic drug, in lipid-based SEDDS using digestion products of long-chain lipids . J Pharm Sci 114 , 103782 ( 2025 ). doi: 10.1016/j.xphs.2025.103782 OpenUrl CrossRef 53. ↵ Yamanouchi , K. et al. Improvement and characterization of oral absorption behavior of clofazimine by SNEDDS: Ǫuantitative evaluation of extensive lymphatic transport . Eur J Pharm Biopharm 187 , 141 – 155 ( 2023 ). doi: 10.1016/j.ejpb.2023.04.009 OpenUrl CrossRef PubMed 54. ↵ Lamb , I. M. , Okoye , I. C. , Mather , M. W. C Vaidya , A. B. Unique Properties of Apicomplexan Mitochondria . Annu Rev Microbiol 77 , 541 – 560 ( 2023 ). doi: 10.1146/annurev-micro-032421-120540 OpenUrl CrossRef PubMed 55. ↵ Lin , S. S. , Gross , U. C Bohne , W. Two internal type II NADH dehydrogenases of Toxoplasma gondii are both required for optimal tachyzoite growth . Mol Microbiol 82 , 209 – 221 ( 2011 ). doi: 10.1111/j.1365-2958.2011.07807.x OpenUrl CrossRef PubMed 56. ↵ Boysen , K. E. C Matuschewski , K. Arrested oocyst maturation in Plasmodium parasites lacking type II NADH:ubiquinone dehydrogenase . J Biol Chem 286 , 32661 – 32671 ( 2011 ). doi: 10.1074/jbc.M111.269399 OpenUrl Abstract / FREE Full Text 57. ↵ Ke , H. et al. Mitochondrial type II NADH dehydrogenase of Plasmodium falciparum (PfNDH2) is dispensable in the asexual blood stages . PLoS One 14 , e0214023 ( 2019 ). doi: 10.1371/journal.pone.0214023 OpenUrl CrossRef PubMed 58. ↵ Guerin , A. et al. Cryptosporidium uses multiple distinct secretory organelles to interact with and modify its host cell . Cell Host Microbe 31 , 650 – 664 e656 ( 2023 ). doi: 10.1016/j.chom.2023.03.001 OpenUrl CrossRef PubMed 59. ↵ Smith , H. B. et al. Listeria monocytogenes requires DHNA-dependent intracellular redox homeostasis facilitated by Ndh2 for survival and virulence . Infect Immun G1 , e0002223 ( 2023 ). doi: 10.1128/iai.00022-23 OpenUrl CrossRef 60. ↵ Maradana , M. R. et al. Dietary environmental factors shape the immune defense against Cryptosporidium infection . Cell Host Microbe 31 , 2038 – 2050 e2034 ( 2023 ). doi: 10.1016/j.chom.2023.11.008 OpenUrl CrossRef 61. Marzook , N. B. et al. The essential host genome for Cryptosporidium survival exposes metabolic dependencies that can be leveraged for treatment . Cell ( 2025 ). doi: 10.1016/j.cell.2025.07.001 OpenUrl CrossRef 62. Funkhouser-Jones , L. J. et al. Microbiota-produced indole metabolites disrupt mitochondrial function and inhibit Cryptosporidium parvum growth . Cell Rep 42 , 112680 ( 2023 ). doi: 10.1016/j.celrep.2023.112680 OpenUrl CrossRef PubMed 63. ↵ Huang , W. et al. Cryptosporidium parvum multidrug resistance protein confers resistance to toxic gut microbial metabolite . Cell Host Microbe 33 , 1589 – 1605 e1589 ( 2025 ). doi: 10.1016/j.chom.2025.07.024 OpenUrl CrossRef PubMed 64. ↵ Gospodaryov , D. V . Alternative NADH dehydrogenase: A complex I backup, a drug target, and a tool for mitochondrial gene therapy . Biochim Biophys Acta Bioenerg 1866 , 149529 ( 2025 ). doi: 10.1016/j.bbabio.2024.149529 OpenUrl CrossRef PubMed 65. ↵ Weinberg , S. E. C Chandel , N. S. Mitochondria reactive oxygen species signaling-dependent immune responses in macrophages and T cells . Immunity 58 , 1904 – 1921 ( 2025 ). doi: 10.1016/j.immuni.2025.07.012 OpenUrl CrossRef PubMed 66. ↵ Ganesan , K. et al. A genetically hard-wired metabolic transcriptome in Plasmodium falciparum fails to mount protective responses to lethal antifolates . PLoS Pathog 4 , e1000214 ( 2008 ). doi: 10.1371/journal.ppat.1000214 OpenUrl CrossRef PubMed 67. ↵ Walzer , K. A. et al. Transcriptional control of the Cryptosporidium life cycle . Nature ( 2024 ). doi: 10.1038/s41586-024-07466-1 OpenUrl CrossRef PubMed 68. ↵ Tandel , J. et al. Life cycle progression and sexual development of the apicomplexan parasite Cryptosporidium parvum . Nat Microbiol 4 , 2226 – 2236 ( 2019 ). doi: 10.1038/s41564-019-0539-x OpenUrl CrossRef PubMed 69. ↵ English , E. D. , Guerin , A. , Tandel , J. C Striepen , B. Live imaging of the Cryptosporidium parvum life cycle reveals direct development of male and female gametes from type I meronts . PLoS Biol 20 , e3001604 ( 2022 ). doi: 10.1371/journal.pbio.3001604 OpenUrl CrossRef PubMed 70. ↵ Shaw , S. et al. Genetic crosses reveal genomic loci responsible for virulence in Cryptosporidium parvum infection . Cell Rep 44 , 116315 ( 2025 ). doi: 10.1016/j.celrep.2025.116315 OpenUrl CrossRef PubMed 71. ↵ Pawlowic , M. C. , Vinayak , S. , Sateriale , A. , Brooks , C. F. C Striepen , B. Generating and Maintaining Transgenic Cryptosporidium parvum Parasites . Curr Protoc Microbiol 46 , 20B 22 21 – 20B 22 32 ( 2017 ). doi: 10.1002/cpmc.33 OpenUrl CrossRef 72. ↵ de Paula Baptista , R. , Xiao , R. , Li , Y. , Glenn , T. C. C Kissinger , J. C. New T2T assembly of Cryptosporidium parvum IOWA annotated with reference genome gene identifiers . bioRxiv ( 2023 ). doi: 10.1101/2023.06.13.544219 OpenUrl Abstract / FREE Full Text 73. ↵ Li , H. C Durbin , R. Fast and accurate short read alignment with Burrows-Wheeler transform . Bioinformatics 25 , 1754 – 1760 ( 2009 ). doi: 10.1093/bioinformatics/btp324 OpenUrl CrossRef PubMed Web of Science 74. ↵ McKenna , A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data . Genome Res 20 , 1297 – 1303 ( 2010 ). doi: 10.1101/gr.107524.110 OpenUrl Abstract / FREE Full Text 75. ↵ Mansfeld , B. N. C Grumet , R. ǪTLseqr: An R Package for Bulk Segregant Analysis with Next-Generation Sequencing . Plant Genome 11 ( 2018 ). doi: 10.3835/plantgenome2018.01.0006 OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted October 07, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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