Complete sequencing of the Cryptosporidium suis gp60 gene reveals a novel type of tandem repeats-Implications for surveillance.

Cryptosporidiosis is an infectious enteric disease caused by species (some of them zoonotic) of the genus Cryptosporidium that in many countries are under surveillance. Typing assays critical to the surveillance of cryptosporidiosis typically involve characterization of Cryptosporidium glycoprotein 60 genes (gp60). Here, we characterized the gp60 of Cryptosporidium suis from two samples-a human and a porcine faecal sample-based on which a preliminary typing scheme was developed. A conspicuous feature of the C. suis gp60 was a novel type of tandem repeats located in the 5' end of the gene and that took up 777/1635 bp (48%) of the gene. The C. suis gp60 lacked the classical poly-serine repeats (TCA/TCG/TCT), which is usually subject to major genetic variation, and the length of the tandem repeat made a typing assay incorporating this region based on Sanger sequencing practically unfeasible. We therefore designed a typing assay based on the post-repeat region only and applied it to C. suis-positive samples from suid hosts from Norway, Denmark, and Spain. We were able to distinguish three different subtypes; XXVa-1, XXVa-2, and XXVa-3. Subtype XXVa-1 had a wider geographic distribution than the other subtypes and was also observed in the human sample. We think that the present data will inform future strategies to develop a C. suis typing assay that could be even more informative by including a greater part of the gene, including the tandem repeat region, e.g., by the use of long-read next-generation sequencing.

Trophozoite fitness dictates the intestinal epithelial cell response to Giardia intestinalis infection.

Giardia intestinalis is a non-invasive, protozoan parasite infecting the upper small intestine of most mammals. Symptomatic infections cause the diarrhoeal disease giardiasis in humans and animals, but at least half of the infections are asymptomatic. However, the molecular underpinnings of these different outcomes of the infection are still poorly defined. Here, we studied the early transcriptional response to G. intestinalis trophozoites, the disease-causing life-cycle stage, in human enteroid-derived, 2-dimensional intestinal epithelial cell (IEC) monolayers. Trophozoites preconditioned in media that maximise parasite fitness triggered only neglectable inflammatory transcription in the IECs during the first hours of co-incubation. By sharp contrast, "non-fit" or lysed trophozoites induced a vigorous IEC transcriptional response, including high up-regulation of many inflammatory cytokines and chemokines. Furthermore, "fit" trophozoites could even suppress the stimulatory effect of lysed trophozoites in mixed infections, suggesting active G. intestinalis suppression of the IEC response. By dual-species RNA-sequencing, we defined the IEC and G. intestinalis gene expression programs associated with these differential outcomes of the infection. Taken together, our results inform on how G. intestinalis infection can lead to such highly variable effects on the host, and pinpoints trophozoite fitness as a key determinant of the IEC response to this common parasite.

Dual RNA Sequencing Reveals Key Events When Different Giardia Life Cycle Stages Interact With Human Intestinal Epithelial Cells In Vitro.

Giardia intestinalis is a protozoan parasite causing diarrheal disease, giardiasis, after extracellular infection of humans and other mammals' intestinal epithelial cells (IECs) of the upper small intestine. The parasite has two main life cycle stages: replicative trophozoites and transmissive cysts. Differentiating parasites (encysting cells) and trophozoites have recently been shown to be present in the same regions of the upper small intestine, whereas most mature cysts are found further down in the intestinal system. To learn more about host-parasite interactions during Giardia infections, we used an in vitro model of the parasite's interaction with host IECs (differentiated Caco-2 cells) and Giardia WB trophozoites, early encysting cells (7 h), and cysts. Dual RNA sequencing (Dual RNAseq) was used to identify differentially expressed genes (DEGs) in both Giardia and the IECs, which might relate to establishing infection and disease induction. In the human cells, the largest gene expression changes were found in immune and MAPK signaling, transcriptional regulation, apoptosis, cholesterol metabolism and oxidative stress. The different life cycle stages of Giardia induced a core of similar DEGs but at different levels and there are many life cycle stage-specific DEGs. The metabolic protein PCK1, the transcription factors HES7, HEY1 and JUN, the peptide hormone CCK and the mucins MUC2 and MUC5A are up-regulated in the IECs by trophozoites but not cysts. Cysts specifically induce the chemokines CCL4L2, CCL5 and CXCL5, the signaling protein TRKA and the anti-bacterial protein WFDC12. The parasite, in turn, up-regulated a large number of hypothetical genes, high cysteine membrane proteins (HCMPs) and oxidative stress response genes. Early encysting cells have unique DEGs compared to trophozoites (e.g. several uniquely up-regulated HCMPs) and interaction of these cells with IECs affected the encystation process. Our data show that different life cycle stages of Giardia induce different gene expression responses in the host cells and that the IECs in turn differentially affect the gene expression in trophozoites and early encysting cells. This life cycle stage-specific host-parasite cross-talk is an important aspect to consider during further studies of Giardia's molecular pathogenesis.

High-Definition DIC Imaging Uncovers Transient Stages of Pathogen Infection Cycles on the Surface of Human Adult Stem Cell-Derived Intestinal Epithelium.

Interactions between individual pathogenic microbes and host tissues involve fast and dynamic processes that ultimately impact the outcome of infection. Using live-cell microscopy, these dynamics can be visualized to study, e.g., microbe motility, binding and invasion of host cells, and intrahost-cell survival. Such methodology typically employs confocal imaging of fluorescent tags in tumor-derived cell line infections on glass. This allows high-definition imaging but poorly reflects the host tissue's physiological architecture and may result in artifacts. We developed a method for live-cell imaging of microbial infection dynamics on human adult stem cell-derived intestinal epithelial cell (IEC) layers. These IEC layers are grown in apical imaging chambers, optimized for physiological cell arrangement and fast, but gentle, differential interference contrast (DIC) imaging. This allows subsecond visualization of both microbial and epithelial surface ultrastructure at high resolution without using fluorescent reporters. We employed this technology to probe the behavior of two model pathogens, Salmonella enterica serovar Typhimurium and Giardia intestinalis, at the intestinal epithelial surface. Our results reveal pathogen-specific swimming patterns on the epithelium and show that Salmonella lingers on the IEC surface for prolonged periods before host cell invasion, while Giardia uses circular swimming with intermittent attachments to scout for stable adhesion sites. The method even permits tracking of individual Giardia flagella, demonstrating that active flagellar beating and attachment to the IEC surface are not mutually exclusive. This work describes a generalizable and relatively inexpensive approach to resolving dynamic pathogen-IEC layer interactions, applicable even to genetically nontractable microorganisms. IMPORTANCE Knowledge of dynamic niche-specific interactions between single microbes and host cells is essential to understand infectious disease progression. However, advances in this field have been hampered by the inherent conflict between the technical requirements for high-resolution live-cell imaging on the one hand and conditions that best mimic physiological infection niche parameters on the other. Toward bridging this divide, we present a methodology for differential interference contrast (DIC) imaging of pathogen interactions at the apical surface of enteroid-derived intestinal epithelia, providing both high spatial and temporal resolution. This alleviates the need for fluorescent reporters in live-cell imaging and provides dynamic information about microbe interactions with a nontransformed, confluent, polarized, and microvilliated human gut epithelium. Using this methodology, we uncover previously unrecognized stages of Salmonella and Giardia infection cycles at the epithelial surface.

Transcriptomic and proteomic analyses of Giardia intestinalis: Intestinal epithelial cell interactions.

Giardia intestinalis is a unicellular protozoan parasite that infects the small intestines of humans and animals. Giardiasis, the disease caused by the parasite, occurs globally across socioeconomic boundaries but is mainly endemic in developing countries and particularly within young children, where pronounced effects manifests in a failure to thrive condition. The molecular pathogenesis of Giardia has been studied using in vitro models of human and rat intestinal epithelial cells (IECs) and parasites from the two major human genotypes or assemblages (A and B). High-quality, genome sequencing of representative isolates from assemblages A (WB) and B (GS) has enabled exploration of these host-parasite models using 'omics' technologies, allowing deep and quantitative analyses of global gene expression changes in IECs and parasites during their interactions, cross-talk and competition. These include a major up-regulation of immune-related genes in the IECs early after the start of interactions, as well as competition between host cells and parasites for nutrients like sugars, amino acids and lipids, which is also reflected in their secretome interactions. Unique parasite proteins dominate these interactions, with many major up-regulated genes being either hypothetical proteins or members of Giardia-specific gene families like the high-cysteine-rich membrane proteins (HCMPs), variable surface proteins (VSPs), alpha-giardins and cysteine proteases. Furthermore, these proteins also dominate in the secretomes, suggesting that they are important virulence factors in Giardia and crucial molecular effectors at the host-parasite interface.