Transmission of Donor-Derived Trypanosoma cruzi and Subsequent Development of Chagas Disease in a Lung Transplant Recipient.

Donor infection status should be considered when accepting an organ for transplant. Here we present a case of Chagas disease developing after a lung transplant where the donor was known to be Trypanosoma cruzi antibody positive. The recipient developed acute Trypanosoma cruzi infection with reactivation after treatment. Chagas disease-positive donors are likely to be encountered in the United States; donor targeted screening is needed to guide decisions regarding organ transplant and posttransplant monitoring.

Donor-derived Trypanosoma cruzi infection in solid organ recipients in the United States, 2001-2011.

Although Trypanosoma cruzi, the parasite that causes Chagas disease, can be transmitted via organ transplantation, liver and kidney transplantation from infected donors may be feasible. We describe the outcomes of 32 transplant recipients who received organs from 14 T. cruzi seropositive donors in the United States from 2001 to 2011. Transmission was confirmed in 9 recipients from 6 donors, including 3 of 4 (75%) heart transplant recipients, 2 of 10 (20%) liver recipients and 2 of 15 (13%) kidney recipients. Recommended monitoring posttransplant consisted of regular testing by PCR, hemoculture, and serology. Thirteen recipients had no or incomplete monitoring; transmission was confirmed in five of these recipients. Four of the five recipients had symptomatic disease and all four died although death was directly related to Chagas disease in only one. Nineteen recipients had partial or complete monitoring for T. cruzi infection with weekly testing by PCR, hemoculture and serology; transmission was confirmed in 4 of 19 recipients with no cases of symptomatic disease. Our results suggest that liver and kidney transplantation from T. cruzi seropositive donors may be feasible when the recommended monitoring schedule for T. cruzi infection is followed and prompt therapy with benznidazole can be administered.

Evidence for separation of HCV subtype 1a into two distinct clades.

The nucleotide sequence diversity present among hepatitis C virus (HCV) isolates allows rapid adjustment to exterior forces including host immunity and drug therapy. This viral response reflects a combination of a high rate of replication together with an error-prone RNA-dependent RNA polymerase, providing for the selection and proliferation of the viruses with the highest fitness. We examined HCV subtype 1a whole-genome sequences to identify positions contributing to genotypic and phenotypic diversity. Phylogenetic tree reconstructions showed two distinct clades existing within the 1a subtype with each clade having a star-like tree topology and lacking definite correlation between time or place of isolation and phylogeny. Identification of significant phylogenetically informative sites at the nucleotide level revealed positions not only contributing to clade differentiation, but which are located at or proximal to codons associated with resistance to protease inhibitors (NS3 Q41) or polymerase inhibitors (NS5B S368). Synonymous/nonsynonymous substitution mutation analyses revealed that the majority of nucleotide mutations yielded synonymous amino acids, indicating the presence of purifying selection pressure across the polyprotein with pockets of positive selection also being detected. Despite evidence for divergence at several loci, certain 1a characteristics were preserved including the length of the alternative reading frame/F protein (ARF/F) gene, and a subtype 1a-specific phosphorylation site in NS5A (S349). Our analysis suggests that there may be strain-specific differences in the development of antiviral resistance to viruses infecting patients who are dependent on the genetic variation separating these two clades.

Development of pilus organelle subassemblies in vitro depends on chaperone uncapping of a beta zipper.

The major subassemblies of virulence-associated P pili, the pilus rod (comprised of PapA) and tip fibrillum (comprised of PapE), were reconstituted from purified chaperone-subunit complexes in vitro. Subunits are held in assembly-competent conformations in chaperone-subunit complexes prior to their assembly into mature pili. The PapD chaperone binds, in part, to a conserved motif present at the C terminus of the subunits via a beta zippering interaction. Amino acid residues in this conserved motif were also found to be essential for subunit-subunit interactions necessary for the formation of pili, thus revealing a molecular mechanism whereby the PapD chaperone may prevent premature subunit-subunit interactions in the periplasm. Uncapping of the chaperone-protected C terminus of PapA and PapE was mimicked in vitro by freeze-thaw techniques and resulted in the formation of pilus rods and tip fibrillae, respectively. A mutation in the leading edge of the beta zipper of PapA produces pilus rods with an altered helical symmetry and azimuthal disorder. This change in the number of subunits per turn of the helix most likely reflects involvement of the leading edge of the beta zipper in forming a right-handed helical cylinder. Organelle development is a fundamental process in all living cells, and these studies shed new light on how immunoglobulin-like chaperones govern the formation of virulence-associated organelles in pathogenic bacteria.

PapG adhesin from E. coli J96 recognizes the same saccharide epitope when present on whole bacteria and as isolated protein.

Purified PapG adhesin from the genetically well-defined uropathogenic Escherichia coli strain J96, as well as whole bacteria, were bound to microtiter plates that carried covalently bound globotetraose and galabiose. The binding was inhibited by soluble saccharide derivatives corresponding to the glycolipids, including all di-, tri-, tetra-, and pentasaccharide fragments of the Forssman antigen and all monodeoxy analogues of galabiose. Analysis of the inhibition pattern showed no significant difference between purified adhesin and whole bacteria. The glucose unit at the reducing end of the natural saccharides was detrimental to PapG binding since deletion of the glucose unit increased the inhibitory power 10-20 fold. The five hydroxyl groups HO-6, -2', -3', -4', -6' of the galabiose unit were shown to be important for PapG binding, presumably via intermolecular hydrogen bonds.

Structural requirements for the glycolipid receptor of human uropathogenic Escherichia coli.

The binding of uropathogenic Escherichia coli to the globo series of glycolipids via P pili is a critical step in the infectious process that is mediated by a human-specific PapG adhesin. Three classes of PapG adhesins exist with different binding specificities to Gal alpha 4Gal-containing glycolipids. The structural basis for PapG recognition of the human glycolipid receptor globoside was investigated by using soluble saccharide analogues as inhibitors of bacterial haemagglutination. The minimum binding epitope was confirmed as the Gal alpha 4Gal moiety, but parts of the GalNAc beta and glucose residues, which flank the Gal alpha 4Gal in globoside (GbO4), were also shown to be important for strong binding. Furthermore, the same five hydroxyl groups of Gal alpha 4Gal in globotriasyl ceramide that were recognized by a previously characterized PapG variant were also recognized by the human-specific PapG in binding the GbO4 that dominates in the human kidney. Saccharide analogues that blocked haemagglutination also blocked the adherence of human uropathogenic E. coli to human kidney sections. Knowledge of the molecular details of the PapG-GbO4 interaction will make it possible to design antiadherence therapeutics.

PapD chaperone function in pilus biogenesis depends on oxidant and chaperone-like activities of DsbA.

Adhesive P pili of uropathogenic Escherichia coli were not assembled by a strain that lacks the periplasmic disulfide isomerase DsbA. This defect was mostly attributed to the immunoglobulin-like pilus chaperone PapD, which possesses an unusual intrasheet disulfide bond between the last two beta-strands of its CD4-like carboxyl-terminal domain. The DsbA-dependent formation of this disulfide bond was critical for PapD's proper folding in vivo. Interestingly, the absence of the disulfide bond did not prevent PapD from folding in vitro or from forming a complex with the pilus adhesin in vitro. We suggest that DsbA maintains nascently translocated PapD in a folding-competent conformation prior to catalyzing disulfide bond formation, acting both as an oxidant and in a chaperone-like fashion. Disulfide bond formation in pilus subunits was also mediated by DsbA even in the absence of PapD. However, the ability of pilus subunits to achieve native-like conformations in vivo depended on PapD. These results suggest that a productive folding pathway for subunits requires sequential interactions with DsbA and the PapD chaperone.

Stable fiber-forming and nonfiber-forming chaperone-subunit complexes in pilus biogenesis.

The P pilus is a composite fiber consisting of a thin adhesive tip fibrillum joined to the pilus rod that mediates specific adherence of uropathogenic Escherichia coli to human uroepithelial cells via the PapG tip adhesin. P pilus assembly depends upon the periplasmic chaperone PapD. The interaction of PapD with different pilus subunits was investigated to gain further insight into pilus assembly. PapA, the major subunit of the pilus rod, formed two periplasmic complexes (DA2 and DA) with PapD. PapK, an adaptor protein that joins the tip fibrillum to the pilus rod, formed only one complex with PapD (DK). Only "fiber forming" or homopolymeric subunits, PapA in the rod and PapE in the tip fibrillum, were able to form subunit-subunit interactions in the periplasm. Subunits that are present in single or low copy in the pilus (PapK and PapG) did not form periplasmic intersubunit interactions. A pulse-chase analysis revealed that a chaperone-PapA complex is a true periplasmic intermediate in pilus assembly.

Chaperone-assisted self-assembly of pili independent of cellular energy.

Assembly of P pili on the surface of pyelonephritic Escherichia coli proceeds from periplasmic chaperone-subunit complexes. The outer membrane protein PapC, which has been termed a molecular usher, is thought to be the site of assembly, where the chaperone dissociates from the subunits as they are incorporated into the pilus across the outer membrane. The kinetics of assembly and the energy requirements of the "secretion" events at the outer membrane were investigated using a pulse-chase analysis in which preformed labeled periplasmic chaperone-subunit complexes were assembled into pili in synchrony by the induction of PapC. Provided that a sufficient amount of PapC was present and functional in the outer membrane, the incorporation of the major PapA subunit into pili was shown to be completed in less than 5 min. Our results also indicated that the targeting of PapC to the outer membrane may be a rate-limiting factor for pilus assembly. Following the arrival of PapC, the formation of pili seemed to proceed spontaneously and was not sensitive to a pH shift or an inhibitor of the electrochemical gradient across the cytoplasmic membrane. We suggest that the secretion of pili across the outer membrane may be independent of cellular energy and thermodynamically driven.

Outer-membrane PapC molecular usher discriminately recognizes periplasmic chaperone-pilus subunit complexes.

P pili are highly ordered composite structures consisting of thin fibrillar tips joined end-to-end to rigid helical rods. The production of these virulence-associated structures requires a periplasmic chaperone (PapD) and an outer membrane protein (PapC) that is the prototype member of a newly recognized class of proteins that we have named "molecular ushers." Two in vitro assays showed that the preassembly complexes that PapD forms with the three most distal tip fibrillar proteins (PapG, PapF, and PapE) bound to PapC. The relative affinity of each complex for PapC was found to correlate with the final position of the subunit type in the tip fibrillum. In contrast, the complexes PapD forms with the major component of the pilus rod, PapA, or the pilus rod initiating protein, PapK, did not recognize PapC. The in vitro data argue that differential targeting of chaperone-subunit complexes to PapC may be part of a mechanism to ensure the correctly ordered assembly of adhesive composite pili.

Degradation of gonococcal peptidoglycan by granule extract from human neutrophils: demonstration of N-acetylglucosaminidase activity that utilizes peptidoglycan substrates.

The degradation of purified Neisseria gonorrhoeae peptidoglycan (PG) by granule extract derived from normal human polymorphonuclear leukocytes was examined. Hen egg lysozyme-resistant, extensively O-acetylated [3H]PG (O-PG) from strain FA19 and lysozyme-sensitive, non-O-acetylated [14C]PG (non-O-PG) from strain RD5 (each containing label in both glucosamine and muramic acid) were mixed and incubated with granule extract at pHs 4.5, 5.5, and 6.5. The rate of degradation of O-PG was uniformly slower than that of non-O-PG in the same tube, but ultimately, even the O-PG was rendered completely soluble. Molecular-sieve high-performance liquid chromatography revealed that both PGs were degraded by granule extract at the pH values tested to disaccharide peptide monomers and peptide-cross-linked oligomers, reflecting the action of human lysozyme. Of particular interest was the appearance of a peak containing free N-acetylglucosamine which was quite prominent in reaction mixtures at pH 4.5, less prominent at pH 5.5, and not detectable at pH 6.5. Free N-acetylglucosamine was not released from control PG samples at any pH in the absence of granule extract. Treatment of purified gonococcal PG monomers with granule extract at pH 4.5 yielded exclusively free N-acetylglucosamine and muramyl peptides with no N-acetylglucosamine. These data suggest that granule extract contains a previously undescribed pH-dependent N-acetylglucosaminidase with specificity for PG as well as an N-acetylmuramidase activity that degrades O-PG less efficiently than it does non-O-PG.

Microculture plaque assay for human and simian cytomegaloviruses.

The plaque assay for human and simian cytomegaloviruses routinely carried out in 60-mm petri dishes (macrocultures) has been adapted for use in microcultures in flat-bottom 16-mm circular wells of disposable plastic trays. Virus titrations and serum neutralization assays carried out in microcultures yielded reproducible results that were identical to those obtained in macrocultures.