Biology subjects

Funkhouser-Jones, L.

Publications and source records attributed to Funkhouser-Jones, L..

4 recordsLinked to original sources

bioRxiv2025

Independent evolution of holocentric centromeres in an early branching apicomplexan parasite

During its asexual cycle, Cryptosporidium parvum (C. parvum) amplifies through three rounds of nuclear division before undergoing cytokinesis to form infectious merozoites. However, chromosome organization and segregation during nuclear division remain unstudied. Here, we visualized H3-histones, including the centromeric histone H3 (CENH3), telomeres, and centrosomes during mitosis. Nuclear division was accompanied by centrosome duplication and elongated microtubules that spanned the dividing nuclei. Surprisingly, C. parvum centromeres detected by CENH3 showed diffuse staining throughout nuclear division, that overlapped with telomeres in the apical nucleus. Analysis of centromeres based on CENH3 capture and DNA sequencing revealed numerous distinct binding sites scattered across all eight chromosomes, including both coding regions and intergenic regions, that were typified by GA rich repeat sequences. The unique holocentric chromosome structure of C. parvum is unlike the single regional centromeres found in related apicomplexans, suggesting it arose independently from other known examples in plants, insects and worms.

microbiology↗

bioRxiv2024

Mendelian segregation and high recombination rates facilitate genetic analyses in Cryptosporidium parvum

Very little is known about the process of meiosis in the apicomplexan parasite Cryptosporidium despite the essentiality of sex in its life cycle. Most cell lines only support asexual growth of Cryptosporidium parvum (C. parvum), but stem cell derived intestinal epithelial cells grown under air-liquid interface (ALI) conditions support the sexual cycle. To examine chromosomal dynamics during meiosis in C. parvum, we generated two transgenic lines of parasites that were fluorescently tagged with mCherry or GFP on chromosomes 1 or 5, respectively. Infection of ALI cultures or Ifngr1-/- mice with mCherry and GFP parasites produced "yellow" oocysts generated by cross-fertilization. Outcrossed oocysts from the F1 generation were purified and used to infect HCT-8 cultures, and phenotypes of the progeny were observed by microscopy. All possible phenotypes predicted by independent segregation were represented equally ([~]25%) in the population, indicating that C. parvum chromosomes exhibit a Mendelian inheritance pattern. Unexpectedly, the most common pattern observed from the outgrowth of single oocysts included all possible parental and recombinant phenotypes derived from a single meiotic event, suggesting a high rate of crossover. To estimate the frequency of crossover, additional loci on chromosomes 1 and 5 were tagged and used to monitor intrachromosomal crosses in Ifngr1-/- mice. Both chromosomes showed a high frequency of crossover compared to other apicomplexans with map distances (i.e., 1% recombination) of 3-12 kb. Overall, a high recombination rate may explain many unique characteristics observed in Cryptosporidium spp. such as high rates of speciation, wide variation in host range, and rapid evolution of host-specific virulence factors. AUTHOR SUMMARYAlthough sex is essential for the transmission and maintenance of infection of Cryptosporidium, it has been historically challenging to study the process of meiosis in this medically relevant protist. We utilize recent methodological advances such as a specialized in vitro culture system, cell sorting, and the generation of transgenic parasites to cross identical strains of parasites in the absence of selection pressure to identify intrinsic chromosome behavior during meiosis. By specifically examining the phenotypes from the first generation of parasites, we reveal that cross-fertilization frequently occurs in parasite populations, chromosomes segregate in a Mendelian manner, and the rate of crossover is high on Chromosomes 1 and 5. Understanding these baseline meiotic mechanisms is essential for planning and interpreting future genetic studies of Cryptosporidium seeking to identify genes associated with phenotypes of interest.

microbiology↗

bioRxiv2023

Microbiota produced indole metabolites disrupt host cell mitochondrial energy production and inhibit Cryptosporidium parvum growth

Cryptosporidiosis is a leading cause of life-threatening diarrhea in young children in resource-poor settings. Susceptibility rapidly declines with age, associated with changes in the microbiota. To explore microbial influences on susceptibility, we screened 85 microbiota- associated metabolites enriched in the adult gut for their effects on C. parvum growth in vitro. We identified eight inhibitory metabolites in three main classes: secondary bile salts/acids, a vitamin B6 precursor, and indoles. Growth restriction of C. parvum by indoles did not depend on the host aryl hydrocarbon receptor (AhR) pathway. Instead, treatment impaired host mitochondrial function and reduced total cellular ATP, as well as directly reduced the membrane potential in the parasite mitosome, a degenerate mitochondria. Oral administration of indoles, or reconstitution of the gut microbiota with indole producing bacteria, delayed life cycle progression of the parasite in vitro and reduced severity of C. parvum infection in mice. Collectively, these findings indicate that microbiota metabolites contribute to colonization resistance to Cryptosporidium infection.

microbiology↗

bioRxiv2020

Gut metabolites influence susceptibility of neonatal mice to cryptosporidiosis.

The protozoan parasite Cryptosporidium is a leading cause of diarrheal disease in those with compromised or under-developed immune systems, particularly infants and toddlers in resource-poor localities. As an enteric pathogen, Cryptosporidium invades the apical surface of intestinal epithelial cells, where it resides in close proximity to metabolites in the intestinal lumen. However, the effect of gut metabolites on susceptibility to Cryptosporidium infection remains largely unstudied. Here, we first identified which gut metabolites are prevalent in neonatal mice when they are most susceptible to Cryptosporidium parvum infection, and then tested the isolated effects of these metabolites on C. parvum invasion and growth. Our findings demonstrate that medium or long-chain saturated fatty acids inhibit C. parvum growth, while long-chain unsaturated fatty acids enhance C. parvum invasion. The influence of these two classes of metabolites on C. parvum infection likely reflects the streamlined metabolism in C. parvum, which is unable to synthesize fatty acids. Hence, gut metabolites, either from diet or produced by the microbiota, play an important role in the early susceptibility to cryptosporidiosis seen in young animals. ImportanceCryptosporidium occupies a unique intracellular niche that exposes the parasite to both host cell contents and the intestinal lumen, including metabolites from the diet and produced by the microbiota. Both dietary and microbial products change over the course of early development, and could contribute to the changes seen in susceptibility to cryptosporidiosis in humans and mice. Consistent with this model, we show that the immature gut metabolome influenced growth of C. parvum in vitro and may increase susceptibility to infection in young mice. Interestingly, metabolites that significantly altered parasite growth were fatty acids, a class of molecules that Cryptosporidium is unable to synthesize de novo. The enhancing effects of polyunsaturated fatty acids and the inhibitory effects of saturated fatty acids provide further insight into reliance on fatty acid salvage and metabolism of this enteric parasite.

microbiology↗