Cryptosporidium and irritable bowel syndrome

Abstract

Cryptosporidium is an apicomplexan parasite that causes gastrointestinal disease in a wide variety of hosts and is associated with waterborne outbreaks. Nonetheless, the parasite is underdiagnosed. Cryptosporidium has been proposed as an etiological cause of irritable bowel syndrome (IBS) in several studies. However, the exact mechanism of pathogenesis is unknown, and no direct link has been discovered. This review will discuss several parasite-induced modifications, such as immunological, microbiome, and metabolite modifications, as well as their interactions. To summarize, Cryptosporidium causes low inflammation, dysbiosis, and unbalanced metabolism, which leads to a lack of homeostasis in the intestine in a comparable pattern to postinfectious IBS.

INTRODUCTION

Cryptosporidium is an apicomplexan parasite of public and veterinary importance, with a new analysis reporting a 7.6% global infection prevalence[1] that can cause intestinal illness in humans, by Consumption of water or food that has been contaminated with the parasite

CRYPTOSPORIDIUM LIFE CYCLE

The Cryptosporidium life cycle begins with the excystation of sporozoites from the oocyst to the intestinal lumen; afterward, the parasite infects intestinal epithelial cells. Then, the parasite forms a parasitophorous vacuole in the microvillous region of the intestinal brush border.

In order to replicate, the parasite undergoes one of two processes: asexual multiplication or development from the oocyst. Asexual multiplication involves the formation of both micro-and macrogametes (gametogony) within the infected cell and subsequently their fusion through fertilization. Meanwhile, the oocyst is the environmentally resistant stage that assists in the transmission of parasite infection in host feces. The oocyst has four infective sporozoites within the oocyst thick wall.[2] The parasite can be transmitted by several direct and indirect routes. Direct infection can occur through the fecal-oral route from infected hosts. A major route of transmission according to data collected from outbreak reports is water. Meanwhile, a rare route of transmission is through the inhalation of oocysts, especially in immunocompromised patients.[3]

CRYPTOSPORIDIUM SPECIES

Several Cryptosporidium species have been identified as causing human infection; Cryptosporidium hominis and Cryptosporidium parvum cause 90% of human infections.[4] However, other species have been recognized as infectious for humans, including Cryptosporidium meleagridis, Cryptosporidium cuniculus, Cryptosporidium felis, and Cryptosporidium canis. Globally, these species are less commonly associated with human cryptosporidiosis. However, distinguishing different Cryptosporidium species microscopically or clinically is uncommon. C. hominis is mostly associated with diarrhea, nausea, vomiting, and malaise, and C. parvum, C. meleagridis, C. canis, and C. felis are linked solely to diarrhea symptoms. Furthermore, it has been demonstrated that infection with C. hominis is correlated with extraintestinal manifestations such as joint pain, eye discomfort, recurring headaches, and fatigue which were not observed in individuals infected by the parasite C. parvum.[5] Furthermore, variances in the clinical presentations have been documented between various species and subtypes of Cryptosporidium among individuals with HIV infection and children.[6,7]

DIAGNOSIS OF CRYPTOSPORIDIOSIS

In cryptosporidiosis, the oocyst is detected by examining the stool sample under a microscope or by analyzing the antigen or nucleic acid. Staining is the most popular diagnostic method in medical laboratories because it is cost-effective (low reagent cost). The stool sample is stained with acid-fast or fluorescent stains.

Parasite antigen detection in a patient stool sample is commercially available. The advantage of this technique is the large number of samples examined in a short time. Additionally, it has been used more frequently in medical laboratories. However, the test sensitivity varies between 70% and 100%.[8,9,10] Moreover, certain rapid tests exhibit reduced specificity and sensitivity when detecting species other than C. parvum or C. hominis,[11,12] and confirmation of positive reactions is needed. These techniques include enzyme immune assays or immunochromatographic techniques.

However, the use of nucleic acid detection by polymerase chain reaction (PCR) has increased, especially in research laboratories, and offers excellent sensitivity and allows species identification.[13] This technique is based on the amplification of Cryptosporidium-specific genes, and the gene encoding 18S rRNA is the most common targeting gene. For genotyping and species identification, the product of the gene encoding 18S rRNA, an approximately 800 base-pair fragment, is sequenced.[14] Additionally, real-time PCR based on smaller fragments has been described[13] due to the similarity between C. hominis and C. parvum (>96%) at the DNA sequence level.[15] The differentiation of these two species has been achieved through the analysis and sequencing of the gp60 gene amplicon.[16] Multilocus methods have been desirable but have not yet been standardized.[17] Before extracting DNA straight from feces, it is advised to break down the oocysts using bead-beating, freeze-thaw, boiling, or chemical lysis[18,19] and isolated the oocyst by floatation technique before the extraction.

CRYPTOSPORIDIUM AND IRRITABLE BOWEL SYNDROME

The gastrointestinal (GI) illness known as irritable bowel syndrome (IBS) is characterized by unsteady bowel movements and stomach pain or discomfort without any anatomical or biochemical abnormalities. IBS affects approximately 9%–23% of the world’s population.[20] IBS pathophysiology is still unclear. Recently, enteric infection and immune activation have been linked with IBS. After enteric infection, some patients will develop IBS with an acute onset that is called “postinfectious IBS” (PI-IBS). Meanwhile, some prospective studies have found that 3%–36% of patients with enteric infection will develop persistent new IBS symptoms. The severity of the symptoms depends on the infecting organism (viruses, bacteria, or parasites). Interestingly, while comparing different pathogens, protozoal enteritis shows the highest risk for PI-IBS development.[21]

One of the parasite infections that are linked with IBS is Cryptosporidium spp. Cryptosporidium has been found in IBS patients, with the onset of GI symptoms after acute infection of Cryptosporidium. The symptoms persist after recovery and parasite clearance. Moreover, there is a similarity between the symptoms that follow C. parvum infection and those described by IBS patients. This similarity suggests that Cryptosporidium might be a possible cause of PI-IBS.[5,22,23] Symptomatic infection of C. hominis and C. parvum were more likely than nonsymptomatic infection to report long-term sequelae, including nausea, vomiting, stomach pain, and diarrhea during a 2-month follow-up period, although joint pain, tiredness, dizziness, recurring headaches, and eye pain were less prevalent.[24,25] However, the severity of the symptoms varied depending on the species of Cryptosporidium.[23,26]

Mechanisms

Cryptosporidiosis has not been linked directly to IBS, but it induces changes, like immunological, microbiome, and metabolite changes, which might involve in the PI-IBS development. Each of these modifications will be discussed in detail and compared with IBS.

Immune response

Many IBS patients have been discovered to have low-grade immunological activity in the colon, where mast cells and T-cells largely play important roles. IBS patients have more mast cells than healthy controls in the small and large intestines.[27,28,29] Given the diverse T-cell variation in IBS, CD4+ and CD8+ T-cells are also believed to be important players in immune responses.[28,29,30] IBS patients show cytokine imbalances. IBS patients have higher levels of interleukin (IL)-1 mRNA in rectal biopsies, along with higher levels of IL-6, IL-8,[31] and tumor necrosis factor-α in their serum,[32] but lower levels of IL-10 mRNA in sigmoid colon biopsies, compared to controls.[31]

In PI-IBS, Sundin et al.[33] found that CD4+ CD8+ double-positive lymphocyte proportions increased in PI-IBS patients, showing a change in the CD3+ lymphocyte subsets. IBS patients’ colons had higher percentages of β7+-expressing lymphocytes.[33,34] The intestinal mucosa of PI-IBS patients had higher levels of interferon (IFN) and lower levels of IL-10, indicating that the infection may disrupt the Th1/Th2 balance. Consequently, immune system dysregulation is likely a significant contributor to IBS.[35]

IBS may be immunologically classified by increases in mucosal mast cell counts, circulating 7+ T-cells, and activated B-cells. Other observed immunological markers of these disorders show significant variation, but for at least a subset of these individuals, a disturbance of mucosal homeostasis is probably related to illness initiation. Together, these data give credence to an immunological-inflammatory etiopathogenesis of IBS.

IBS and a Cryptosporidium infection have comparable immune responses. Increased expression of the Th1 cytokines IFN and IL-12, as well as IL-18 in mice, has been linked to immunity against C. parvum infection.[36,37] Furthermore, the early phases of infection are more lethal in β7^−/− neonatal mice lacking the integrin α4 β7 necessary for homing of activated mucosal T-cells to the gut.[38] Finally, C. parvum infection induces an increase in toll-like receptor (TLR) 4 and ICAM-1 expression.[39,40] TLR4[41] and ICAM-1 increased expression of these two receptors has been associated with IBS.[42]

In addition, C. parvum infection-induced jejunal hypersensitivity to distension persisted for more than 100 days after the parasites disappeared by themselves in an immunocompetent rat model.[43] Furthermore, Khaldi et al.[44] described jejunal hypersensitivity after Cryptosporidium infection related to activated mast cell accumulation in rats. The extent of infection and long-term postinfectious changes of jejunal sensitivity and activated mast cell proliferation in newborn immunocompetent rats were also predicted to exhibit similar isolate reliance.

Thus, immunological activation during Cryptosporidium infection is similar to IBS patients’ loss of homeostasis, and this immune pattern can be triggered by Cryptosporidiosis. This lack of homeostasis could also be linked to changes in the GI microbiota, which has been linked to IBS.[45]

Microbiome

Numerous microorganisms, including bacteria, viruses, fungi, archaea, and other parasitic species, make up the gut microbiota. This type of microbial community organization is thought to represent individuals and support the metabolism of the host and even the development of organs.[46] There have been reports of bacterial changes in IBS patients, including an increased proportion of the phylum Firmicutes to Bacteroidetes as well as an increase in the abundance of the genus Bacteroides and a decrease in Bifidobacterium,[47] at the genus level. Additionally, gut microorganisms have an impact on IBS symptoms.[48,49] In addition to affecting intestinal ecology and motility, enterotoxigenic Bacteroides fragilis destroys intestinal glycoproteins that cause diarrhea and discomfort.[50,51] In contrast, Bifidobacterium infantis can reduce proinflammatory cytokines while maintaining anti-inflammatory cytokines, therefore alleviating IBS symptoms.[52] The function of TLRs as a point of contact for the immune system, the gut microbiota, and IBS in addition, microbiota might effect IBS by change the expression of Toll like receptors.[53,54] In a recent study, Jalanka et al.[41] examined the connection between IBS patients’ gene expression of TLR4 and associated receptors and found evidence that intestinal bacteria are likely to be the cause of the low inflammation observed in people with IBS.

Although there are many different microbes in the intestinal microbiota, it appears that microbe-association pattern recognition is crucial in directing immune responses. Microbial dysbiosis has been demonstrated to induce inflammation[55] and disrupt normal lymphocyte function in PI-IBS,[56] eventually sustaining chronic, low-grade inflammation involving bacteria, viruses, fungi, archaea, and other parasite organisms. These results indicate that the presence of gut infection has a direct effect on the gut microbiome.

During Cryptosporidium infection, the gut microbiota is altered. As shown by Rahman et al. 2022,[57] a decrease in the diversity of fecal microbiota in neonatal calves is linked to a higher excretion rate of Cryptosporidium spp., while conversely, age has the opposite effect. The proportion of Lactobacillus, Bacteroides, Akkermansia, Desulfovibrio, Prevotella, and Helicobacter was correlated with C. parvum.[57] In negative binomial models of Cryptosporidium, the four genera (Ruminiclostridium, Alistipes, Parasutterella, and Faecalibacterium) connected to Cryptosporidium spp. infection declined as more oocysts were found.[58]

Cryptosporidium infection modifies the gut microbiota. Carey et al.[59] discovered that children with cryptosporidiosis experienced diarrheal symptoms when Megasphaera were at a reduced level. Additionally, they discovered that these children’s microbiomes were suggestive of diarrhea both before and after Cryptosporidium spp. infection. While, mice infected with Cryptosporidium spp. had higher relative abundances of Bacteroidetes and lower abundances of Firmicutes.[60] Karpe et al. showed[61] the abundance of Faecalibaculum, Barnesiella, and Lactobacillus in the small intestine, cecum, and colon when mice were infected with C. parvum. Additionally, it was shown that populations of Lactobacillus, Lachnospiraceae, Desulfovibrio, and Coriobacterium grew, particularly in the jejunum and ileum, whereas those of Faecalibaculum and Lachnospiraceae gradually declined from the duodenum onward. Except for Lactobacillus, the numbers of Coriobacteriaceae, Ruminococcaceae, and Lachnospiraceae species in the feces grew. Moreover, Cryptosporidium muris-infected mice have a microbiome rich in bacteria that produce short-chain fatty acids, like Lachnospiraceae and Prevotella.[62] However, C. parvum-induced microbiota disruption appeared to be reversible.[63]

Despite the variability in the results obtained from different animals, these findings suggest that some bacterial species may find the gut bacterial ecology less favorable when Cryptosporidium spp. are present, which suggests that immune activation and subsequent microbiota change may be the cause of the potential link between Cryptosporidium and IBS.

Metabolomics

Intestinal metabolites are chemicals that are produced by intestinal cells in response to food. These metabolites can have a significant impact on the health of the individual. For example, butyrate is an anti-inflammatory molecule.[64,65] C. parvum decreased the abundance of butyrate-producing pathways in bacteria in infected goats.[66] While, in mice infected with C. parvum, Butyrate levels were increased in the intestine.[61] Additionally, the study discovered that glutamine/glutamate metabolism was upregulated in the infected cecum, glutamine is involved in the energy supply to the gut’s epithelial cells, and glutamine deficiency might contribute to IBS symptoms, and taking dietary supplements of glutamine can improve intestinal permeability and lessen IBS with diarrhea (IBS-D) symptoms.[67] Proteins involved in the production of branched-chain amino acids, glutamate, glycine, and alanine were downregulated, according to proteomic studies.[39] This study in proteomics aimed to analyze the alterations in the proteome caused by C. parvum infection in HCT-8 cells as compared to uninfected cells. The findings revealed a decrease in proteins involved in steroid production and fatty acid metabolism.[39] The study also found a decrease in proteins involved in the pathway for the metabolism of alpha-linoleic acid. Linoleic acid alleviates IBS symptoms.[68] It is important to note that Cryptosporidium induce the changes in the short-chain fatty acids through the above-mentioned changes in the microbiota.[61]

Intriguingly, IBS reduces the intestinal absorption of folate. Infection with C. parvum causes downregulation of proteins required in the production of folate. This might explain the symptoms of systemic C. parvum infection. These findings showed how the parasite modifies the host’s metabolic pathway, which may be directly or indirectly responsible for IBS symptoms.

Additionally, at 7 days post infection, metabolites related to metabolic pathways, steroid hormone biosynthesis, and unsaturated fatty acid biosynthesis were upregulated in BALB/c mice infected with C. muris. Dopaminergic synapsis was also upregulated 28 days after infection,[62] suggesting that Cryptosporidium may be involved in the brain–gut axis and that the brain–gut axis might be a contributor to the pathogenesis of IBS.[69] Another apicomplexan parasite, Toxoplasma gondii, has been shown to alter catecholamine production,[70] while this study also demonstrated that the parasite downregulates a protein involved in steroid hormone biosynthesis, which could be another field worth exploring. Understanding the interaction between host and parasite could not only help in understanding how the host physiology has adapted to the parasite as well.

All of these findings suggest that Cryptosporidium alters the metabolome of the host intestine in a manner that resembles the pathophysiology of IBS.

Mucosal permeability

During PI-IBS, the permeability of the intestinal mucosa increases.[71,72,73,74] While IBS-D protein analysis revealed changes in actin-cytoskeleton function and signaling,[75] The transcriptomic investigations revealed alterations in the tight junction and adherens junction signaling pathways.[76,77] Animals infected with C. parvum exhibit a considerable increase in intestinal permeability, reduced transepithelial resistance, and enhanced paracellular permeability once intestinal epithelial cells are infected by the parasite in vivo.[78] The downregulation of several essential epithelial adherens junction and tight junction components is what leads to this disruption of barrier function.[79] Furthermore, the gathering of host filamentous actin in close proximity to the infection site leads to the formation of a pedestal-shaped structure, which is a key aspect of the relationship between the parasite Cryptosporidium and its host.[80] Finally, inflammatory monocytes have a role in the breakdown of intestinal barrier function during cryptosporidiosis.[81]

Extracellular vesicles

EVs contribute to the regulation of epithelial barrier integrity and function, By exerting an influence on the development of functional complexes in intestinal epithelial cells. Extracellular vesicles (EVs) contribute to the regulation of epithelial barrier integrity and function. They also influence the movement and activity of immune cells by controlling cytokine expression and transporting a variety of chemokines and lipids.[82] In IBS patients, the regulation of intestinal permeability is influenced by the overexpression of miR-29a in the blood exosomes of IBS patients.[83] By controlling the G protein-coupled receptors in the GI tract, regulators of G protein signaling (RGS) proteins regulate the activity of the endogenous opioid, cannabinoid, and serotonin systems, contributing to GI inflammation and visceral discomfort.[84] Furthermore, a recent study found that exosomal miR-148b-5p, which is produced from mesenchymal stem cells, mediates the immunosuppressive impact in inflammatory bowel disease by suppressing 15-LOX-1 in macrophages.[85,86]

In the context of patients with IBS, exosomes derived from serum have been found to enhance the permeability of colonic epithelial cells through the signaling pathway involving miR-148b-5p and RGS2.

However, since exosomes might explain the systemic effect on the intestine in mild infection, the direct impact of exosomes released during intestinal infection on the induction of IBS is an area that deserves further investigation. C. parvum infection causes host epithelial cells to produce exosomes by activating TLR4/NF-B signaling.[87] Exosomes that have been secreted contain multiple types of antimicrobial peptides, which exhibit the ability to adhere to C. parvum sporozoites and exert an inhibitory effect against C. parvum. These EVs might have a relevant role in modulating intestinal homeostasis at various levels, as reviewed in Diaz-Garrido et al.’s study.[88] Emerging research indicates that certain microRNA molecules found in feces can play a role in regulating the composition of the gut microbiota. These microRNA molecules are released by enterocytes into the intestinal lumen through EVs. This important finding highlights how both host EVs and miRNAs derived from enterocytes have an influence on the microbial population residing in the gut.

CONCLUSION

Cryptosporidium infection might facilitate the development of IBS by changing the hemostasis between the immune system and microbiota, and the microbiota-to-metabolome association is imbalanced during Cryptosporidium infection, leading to medullated inflammatory cytokine activity and low inflammation in the gut of a host, as summarized in Figure 1.

Figure 1.

A Cryptosporidium infection changes the hemostasis between the immune system and microbiota, leading to low inflammation in the gut and medullated inflammatory cytokine activity, furthermore it increases intestinal permeability and modulates the microbiota-to-metabolome association, INF: INF is released by dendritic cells and monocytes, while IL-18 from cells infected with cryptosporidium prompts the release of INF. This leads to a low grade inflammation that affects the gut microbiota, compounded by the direct existence of cryptosporidium, which releases different compounds from infected cells. These combined factors contribute to increased intestinal permeability and thus lead to the onset of symptoms associated with irritable bowel syndrome

During the infection, C. parvum had a Cryptosporidium secretion mechanism that may transfer parasite proteins into the infected enterocyte, demonstrating particularly how Cryptosporidium can manipulate the host.[89] Nevertheless, it’s intriguing how the parasite could cause these modifications after infection., However as there isn’t enough proof to conclude that Cryptosporidium is eradicated from enterocytes, so these alterations could be the result of the parasite dormant stage, similarly other apicomplexans, such as Plasmodium vivax and Plasmodium ovale, are known to have dormant stages, whereas Toxoplasma infection is lifelong. This could imply that Cryptosporidium has a dormant stage within the host that is undetectable by conventional methods. Although there is no evidence to support this allegation, there is also no evidence to refute it.

However, all the data presented related changes that occur in cell culture and mice during acute infection. However, the chronic phase and postinfection modification were not investigated, not. Except for Karpe et al.[61] who examined metabolites and microbiomes in infected mice on day 28 post infection. Moreover, the results reported in this article were obtained from different Cryptosporidium strains in different hosts. Due to the observed variety of clinical presentations of Cryptosporidium species and subtypes, these experiments should be performed with human-specific Cryptosporidium strains. Because Cryptosporidium causes several changes in the host, it should not be ignored and requires better control and diagnosis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.