Sequence analysis of the HIV-1 protease coding region of 18 HIV-1-infected patients prior to HAART and possible implications on HAART.

BACKGROUND: The majority of HIV-infected patients are treated with highly active antiretroviral therapy (HAART) consisting of a combination of inhibitors of the protease (PIs) and the reverse transcriptase (RTIs). Analysis of mutations within these enzymes which are associated with development of resistance to the applied inhibitors is of major clinical importance. In particular, pre-existing mutations in previously untreated individuals may adversely influence the efficacy of HAART. OBJECTIVES: The sequences of the protease coding regions of 18 HIV-1-infected patients were analysed prior to HAART. STUDY DESIGN: DNA was extracted from whole blood samples of HIV-1-infected treatment-naive patients. The protease coding region was amplified by nested PCR and sequenced directly. The resulting amino acid substitutions were analysed for known mutations associated with known resistance to PIs. RESULTS: In all 18 analysed individuals we found 1-10 amino acid substitutions per patient in their HIV-1 protease coding region. These mutations occurred altogether at 27 positions of the 99 amino acids of the protease coding region. Seven of these mutated positions are associated with described resistance to PIs. Altogether, 15 of the 18 patients (83%) carried at least one such resistance-conferring alteration in their protease coding region. All patients are currently followed up during their present therapy to detect possible resistance formation to the applied PIs. CONCLUSIONS: A large variety of pre-existing mutations associated with resistance to PIs was observed prior to their treatment. As none of the patients ever received HAART before and infection with resistant viral strains is very unlikely, these amino acid substitutions evidently reflect natural polymorphism of the HIV-1 protease coding region.

Identification of a polyketide synthase gene (pksP) of Aspergillus fumigatus involved in conidial pigment biosynthesis and virulence.

Aspergillus fumigatus is an important pathogen of the immunocompromised host causing pneumonia and invasive disseminated disease with high mortality. Previously, we identified a mutant strain (white, W) lacking conidial pigmentation and, in addition, the conidia showed a smooth surface morphology, whereas wild-type (WT) conidia are grey-green and have a typical ornamentation. W conidia appeared to be less protected against killing by the host defence, e.g., were more susceptible to oxidants in vitro and more efficiently damaged by human monocytes in vitro than WT conidia. When compared to the WT, the W mutant strain showed reduced virulence in a murine animal model. Genetic analysis suggested that the W mutant carried a single mutation which caused all of the observed phenotypes. Here. we report the construction of a genomic cosmid library of A. fumigatus and its use for complementation of the W mutant. Transformation of the W mutant was facilitated by co-transformation with plasmid pHELP1 carrying the autonomously replicating ama1 sequence of A. nidulans which also increased the transformation efficiency of A. fumigatus by a factor of 10. Using this cosmid library a putative polyketide synthase gene, designated pksP (polyketide synthase involved in pigment biosynthesis) was isolated. The pksP gene has a size of 6660 bp. pksP consists of five exons separated by short (47-73 bp) introns. Its deduced open reading frame is composed of 2146 amino acids. The pksP gene complemented both the white phenotype and the surface morphology of the W mutant conidia to wild type. Whereas W mutant conidia caused a strong reactive oxygen species (ROS) release by polymorphonuclear leukocytes, the ability of pksP-complemented W mutant conidia to stimulate ROS release was significantly reduced and comparable to that of WT conidia. In addition, the complemented strains showed restored virulence in a mouse model.

Isolation and characterization of a pigmentless-conidium mutant of Aspergillus fumigatus with altered conidial surface and reduced virulence.

Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease with high mortality. The factors contributing to the predominance of A. fumigatus as an opportunistic pathogen are largely unknown. Since the survival of conidia in the host is a prerequisite for establishing disease, we have been attempting to identify factors which are associated with conidia and, simultaneously, important for infection. Therefore, an A. fumigatus mutant strain (white [W]) lacking conidial pigmentation was isolated. Scanning electron microscopy revealed that conidia of the W mutant also differed in their surface morphology from those of the wild type (WT). Mutant (W) and WT conidia were compared with respect to their capacities to stimulate an oxidative response in human phagocytes, their intracellular survival in human monocytes, and virulence in a murine animal model. Luminol-dependent chemiluminescence was 10-fold higher when human neutrophils or monocytes were challenged with W conidia compared with WT conidia. Furthermore, mutant conidia were more susceptible to killing by oxidants in vitro and were more efficiently damaged by human monocytes in vitro than WT conidia. In a murine animal model, the W mutant strain showed reduced virulence compared with the WT. A reversion analysis of the W mutant demonstrated that all phenotypes associated with the W mutant, i.e., altered conidial surface, amount of reactive oxygen species release, susceptibility to hydrogen peroxide, and reduced virulence in an murine animal model, coreverted in revertants which had regained the ability to produce green spores. This finding strongly suggests that the A. fumigatus mutant described here carries a single mutation which caused all of the observed phenotypes. Our results suggest that the conidium pigment or a structural feature related to it contributes to fungal resistance against host defense mechanisms in A. fumigatus infections.