We screened 1200 existing drugs and identified vorinostat as a new lead that displayed outstanding anticryptosporidial efficacy both in vitro and in vivo. We also confirmed that vorinostat was a ...potent inhibitor of histone deacetylase in the parasite.
Abstract
Background
Cryptosporidiosis affects all human populations, but can be much more severe or life-threatening in children and individuals with weak or weakened immune systems. However, current options to treat cryptosporidiosis are limited.
Methods
An in vitro phenotypic screening assay was employed to screen 1200 existing drugs for their anticryptosporidial activity and to determine the inhibitory kinetics of top hits. Selected top hits were further evaluated in mice. The action of the lead compound vorinostat on the parasite histone deacetylase (HDAC) was biochemically validated.
Results
Fifteen compounds exhibited anticryptosporidial activity at nanomolar level in vitro. Among them, the histone deacetylase (HDAC) inhibitor vorinostat retained outstanding efficacy in vitro (half maximal effective concentration, EC50 = 203 nM) and in an interleukin 12 knockout mouse model (50% inhibition dose = 7.5 mg/kg). Vorinostat was effective on various parasite developmental stages and could irreversibly kill the parasite. Vorinostat was highly effective against the parasite native HDAC enzymes (half maximal inhibitory concentration, IC50 = 90.0 nM) and a recombinant Cryptosporidium parvum HDAC (the inhibitor constant, Ki = 123.0 nM).
Conclusions
These findings suggest the potential for repurposing of vorinostat to treat cryptosporidiosis, and imply that the parasite HDAC can be explored for developing more selective anticryptosporidial therapeutics.
The emergence of SARS-CoV-2 variants with mutations in the spike protein is raising concerns about the efficacy of infection- or vaccine-induced antibodies. We compared antibody binding and live ...virus neutralization of sera from naturally infected and Moderna-vaccinated individuals against two SARS-CoV-2 variants: B.1 containing the spike mutation D614G and the emerging B.1.351 variant containing additional spike mutations and deletions. Sera from acutely infected and convalescent COVID-19 patients exhibited a 3-fold reduction in binding antibody titers to the B.1.351 variant receptor-binding domain of the spike protein and a 3.5-fold reduction in neutralizing antibody titers against SARS-CoV-2 B.1.351 variant compared to the B.1 variant. Similar results were seen with sera from Moderna-vaccinated individuals. Despite reduced antibody titers against the B.1.351 variant, sera from infected and vaccinated individuals containing polyclonal antibodies to the spike protein could still neutralize SARS-CoV-2 B.1.351, suggesting that protective humoral immunity may be retained against this variant.
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•Antibodies from infected and vaccinated individuals bind to the B.1.351 RBD•Convalescent sera through eight months can neutralize the B.1.351 variant•Serum from vaccinated individuals retains neutralization against the B.1.351 variant
In this study, Edara et al. (2021) report that, despite reduced antibody binding to the B.1.351 RBD, sera from infected (acute and convalescent) and Moderna (mRNA-1273)-vaccinated individuals were still able to neutralize the SARS-CoV-2 B.1.351 variant, suggesting that protective humoral immunity may be retained against this variant.
Cryptosporidium spp. are opportunistic protozoan parasites that infect epithelial cells of the small intestine, causing diarrheal illness in humans. Differences in severity may be due to the ...immunological status of the host, malnutrition or prior exposure but may also be due to differences in the host gut flora. We examined changes in bacterial flora following antibiotic treatment to determine how cryptosporidial infections and gut integrity were affected by alterations in the microbiome. DNA was extracted from fecal and intestinal samples during peak infection. V4 region amplicons were generated and sequenced using 16sRNA on an Illumina MiSeq. Species evenness and richness were estimated using the Shannon diversity index. There was a significant decrease in anaerobes and overgrowth of Enterobacteriaceae in mice treated with cloxacillin. We also examined levels of short-chain fatty acids in fecal samples. There was a significant decrease in acetate, propionate, and butyrate in these same mice. Concurrent with the shift in bacterial infection was a significant increase in severity of cryptosporidial infection and increase in gut permeability. Treatment with other antibiotics significantly altered the microbiome but did not change the infection, suggesting that specific alterations in the host microbiome allow for more favorable growth of the parasite.
Cryptosporidium spp. are opportunistic protozoan parasites that infect epithelial cells in the intestinal tract and cause a flu-like diarrheal illness. Innate immunity is key to limiting the ...expansion of parasitic stages early in infection. One mechanism in which it does this is through the generation of early cytokines, such as IL-18. The processing and secretion of mature IL-18 (and IL-1β) is mediated by caspase-1 which is activated within an inflammasome following the engagement of inflammasome-initiating sensors. We examined how the absence of caspase-1 and caspase-11, the adapter protein Asc, and other inflammasome components affects susceptibility to cryptosporidial infection by these and other key cytokines in the gut. We found that Casp-11−/−Casp-1−/− knockout mice have increased susceptibility to Cryptosporidium parvum infection as demonstrated by the 35-fold higher oocyst production (at peak infection) compared to wild-type mice. Susceptibility correlated with a lack of IL-18 in caspase-1 and caspase1/11 knockout mice, whereas IL-18 is significantly elevated in wildtype mice. IL-1β was not generated in any significant amount following infection nor was any increased susceptibility observed in IL-1β knockout mice. We also show that the adapter protein Asc is important to susceptibility, and that the caspase-1 canonical inflammasome signaling pathway is the dominant pathway in C. parvum resistance.
Auranofin is an FDA-approved gold-containing compound used for the treatment of rheumatoid arthritis. Recent reports of antimicrobial activity against protozoa and bacteria indicate that auranofin ...targets the reductive enzyme thioredoxin reductase (TrxR). We evaluated auranofin as well as five auranofin analogs containing N-heterocyclic carbenes (instead of the triethylphosphane present in auranofin) and five gold-carbene controls for their ability to inhibit or kill Helicobacter pylori in vitro. Auranofin completely inhibited bacterial growth at 1.2 μM. Purified H. pylori TrxR was inhibited by auranofin in a cell-free assay (IC50 ∼88 nM). The most active gold(I)-N-heterocyclic carbene compounds exhibited MICs comparable to auranofin against H. pylori (2 μM), while also exhibiting lower toxicities for human embryonic kidney cells (HEK-293T cells). Median toxic concentrations (TC50) were 13–20-fold higher compared to auranofin indicating that they were less cytotoxic. The N-heterocyclic carbene analogs maybe well tolerated, but further evaluation is needed in vivo. Finally, auranofin was synergistic with the antibiotic amoxicillin, suggesting that targeting both the reductive enzyme TrxR and cell wall synthesis may be effective against H. pylori infections.
This article describes the antimicrobial activity of auranofin and N-heterocyclic carbene gold-analogs against Helicobacter pylori.
IL-18 is known to play a key role limiting Cryptosporidium parvum infection. In this study, we show that IL-18 depletion in SCID mice significantly exacerbates C. parvum infection, whereas, treatment ...with recombinant IL-18 (rIL-18), significantly decreases the parasite load, as compared to controls. Increases in serum IFN-γ levels as well as the up-regulation of the antimicrobial peptides, cathelicidin antimicrobial peptide and beta defensin 3 (Defb3) were observed in the intestinal mucosa of mice treated with rIL-18. In addition, C. parvum infection significantly increased mRNA expression levels (> 50 fold) of the alpha defensins, Defa3 and 5, respectively. Interestingly, we also found a decrease in mRNA expression of IL-33 (a recently identified cytokine in the same family as IL-18) in the small intestinal tissue from mice treated with rIL-18. In comparison, the respective genes were induced by IL-18 depletion. Our findings suggest that IL-18 can mediate its protective effects via different routes such as IFN-γ induction or by directly stimulating intestinal epithelial cells to increase antimicrobial activity.
Highlights • Depletion of DCs in vivo increases susceptibility to Cryptosporidium parvum infection. • Adoptive transfer of stimulated DCs reduces C. parvum infection levels. • Reduction in infection ...correlates to an increase in IFN-γ in T cell. • DCs pulsed with sporozoites and co-cultured with CD4+ and CD8+ T cells produced higher IFN-γ levels.
Cryptosporidium parvum is a protozoan parasite that infects the epithelial cells of the small intestine causing diarrheal illness in humans. While T cells are known to be important in resistance and ...recovery from infection, little has been characterized as to the phenotypic expression of surface effector and memory markers after infection. We used an acute model of infection (C57BL/6 interleukin-12p40), which develops long-standing resistance to re-infection, to characterize expression of different effector and memory cells. Using flow cytometry, we found that heterogeneous populations were generated after infection, consisting of both CD62Lhigh central memory T cells (TCM) and CD62Llow effector memory T cells (TEM) that were competent to produce the Th type 1 effector cytokine, IFN-γ. Both CD4+ and CD8+ TCM and TEM populations persisted in the absence of infection (up to 60 days post-infection). Additionally, transfer of either CD62LlowCD4+ TEM or CD62LhighCD4+ TCM into naive recipients resulted in a protective response. Taken together, these studies show that distinct subsets of effector and memory CD4+ T cells develop after infection with C. parvum, and mediate protective immunity to re-challenge.
Background. Cryptosporidium is emerging as 1 of the 4 leading diarrheal pathogens in children in developing countries. Its infections in patients with AIDS can be fatal, whereas fully effective ...treatments are unavailable. The major goal of this study is to explore parasite fatty acyl-coenzyme A synthetase (ACS) as a novel drug target. Methods. A colorimetric assay was developed to evaluate biochemical features and inhibitory kinetics of Cryptosporidium parvum ACSs using recombinant proteins. Anticryptosporidial efficacies of the ACS inhibitor triacsin C were evaluated both in vitro and in vivo. Results. Cryptosporidium ACSs displayed substrate preference toward long-chain fatty acids. The activity of parasite ACSs could be specifically inhibited by triacsin C with the inhibition constant Ki in the nanomolar range. Triacsin C was highly effective against C. parvum growth in vitro (median inhibitory concentration, 136 nmol/L). Most importantly, triacsin C effectively reduced parasite oocyst production up to 88.1% with no apparent toxicity when administered to Cryptosporidium-infected interleukin 12 knockout mice at 8-15 mg/kg/d for 1 week. Conclusions. The findings of this study not only validated Cryptosporidium ACS (and related acyl-acyl-carrierprotein-ligases) as pharmacological targets but also indicate that triacsin C and analogues can be explored as potential new therapeutics against the virtually untreatable cryptosporidial infection in immunocompromised patients.
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