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Biliary cryptosporidiosis is associated with acquired immunodeficiency syndrome (AIDS) cholangiopathy and occurs almost exclusively in adult patients with AIDS. Infection of biliary epithelial cells (cholangiocytes) with Cryptosporidium parvum induces Toll-like receptor (TLR) 4 expression and stimulates a TLR-dependent response against infection. Here, we tested whether human immunodeficiency virus type 1 (HIV-1) Tat affects TLR expression and, hence, anti-C. parvum defense responses. Using an in vitro model of human biliary cryptosporidiosis, we found that recombinant Tat protein increased TLR4 mRNA expression in both uninfected and C. parvum-infected cholangiocytes. Conversely, Tat decreased TLR4 protein levels and suppressed C. parvum-induced TLR4 protein expression. Using actinomycin to inhibit transcription, we found that Tat increased the half-life of TLR4 mRNA from approximately 25 to 60 min, and RNA gel-shift assays demonstrated direct binding of Tat to TLR4 mRNA. In vitro transcription/translation studies suggested that Tat does not affect transcription but does decrease TLR4 translation. Importantly, more parasites were found in Tat-treated cells than in control cells 48 h after infection. These findings suggest that Tat inhibits cholangiocyte TLR4 protein expression through translational inhibition. These events appear to diminish the ability of cholangiocytes to initiate an innate immune response to C. parvum. We suggest that these findings may contribute to the unusual susceptibility of HIV-infected individuals to biliary cryptosporidiosis.

Frataxin is a mitochondrial protein structurally conserved from bacteria to humans. Eukaryotic frataxins are known to be involved in the maintenance of mitochondrial iron balance via roles in iron delivery and iron detoxification. The prokaryotic frataxin homolog, CyaY, has been shown to bind and donate iron for the assembly of [2Fe-2S] clusters in vitro. However, in contrast to the severe phenotypes associated with the partial or complete loss of frataxin in humans and other eukaryotes, deletion of the cyaY gene does not cause any obvious alteration of iron balance in bacterial cells, an effect that probably reflects functional redundancy between CyaY and other bacterial proteins. To study CyaY function in a nonredundant setting, we have expressed a mitochondria-targeted form of CyaY in a Saccharomyces cerevisiae strain depleted of the endogenous yeast frataxin protein (yfh1Delta). We show that in this strain CyaY complements to a large extent the loss of iron-sulfur cluster enzyme activities and heme synthesis, and thereby maintains a nearly normal respiratory growth. In addition, CyaY effectively protects yfh1Delta from oxidative damage during treatment with hydrogen peroxide but is less efficient in detoxifying excess labile iron during aerobic growth.