Maraviroc Population Pharmacokinetics Within the First 6 Weeks of Life.

BACKGROUND: Treatment and prophylaxis options for neonatal HIV are limited. This study aimed to develop a population pharmacokinetic model to characterize the disposition of maraviroc in neonates to inform dosing regimens and expand available options. METHODS: Using maraviroc concentrations from neonates who received either a single dose or multiple doses of 8 mg/kg of maraviroc in the first 6 weeks of life, a population pharmacokinetic model was developed to determine the effects of age, sex, maternal efavirenz exposure and concomitant ARV therapy on maraviroc disposition. The final model was used in Monte Carlo simulations to generate expected exposures with recommended dosing regimens. RESULTS: A total of 396 maraviroc concentrations, collected in the first 4 days of life, at 1 week, at 4 weeks and at 6 weeks, from 44 neonates were included in the analysis. After allometrically scaling for weight, age less than 4 days was associated with a 44% decreased apparent clearance compared with participants 7 days to 6 weeks of life. There were no differences identified in apparent clearance or volume of distribution from ages 7 days to 6 weeks, sex, maternal efavirenz exposure or concomitant nevirapine therapy. Monte Carlo simulations with FDA-approved weight band dosing resulted in the majority of simulated patients (84.3%) achieving an average concentration of ≥75 ng/mL. CONCLUSIONS: While maraviroc apparent clearance is decreased in the first few days of life, the current FDA-approved maraviroc weight band dosing provides maraviroc exposures for neonates in the first 6 weeks of life, which were consistent with adult maraviroc exposure range. Maraviroc provides another antiretroviral treatment option for very young infants.

Plasma membrane localization is required for RasA-mediated polarized morphogenesis and virulence of Aspergillus fumigatus.

Ras is a highly conserved GTPase protein that is essential for proper polarized morphogenesis of filamentous fungi. Localization of Ras proteins to the plasma membrane and endomembranes through posttranslational addition of farnesyl and palmitoyl residues is an important mechanism through which cells provide specificity to Ras signal output. Although the Aspergillus fumigatus RasA protein is known to be a major regulator of growth and development, the membrane distribution of RasA during polarized morphogenesis and the role of properly localized Ras signaling in virulence of a pathogenic mold remain unknown. Here we demonstrate that Aspergillus fumigatus RasA localizes primarily to the plasma membrane of actively growing hyphae. We show that treatment with the palmitoylation inhibitor 2-bromopalmitate disrupts normal RasA plasma membrane association and decreases hyphal growth. Targeted mutations of the highly conserved RasA palmitoylation motif also mislocalized RasA from the plasma membrane and led to severe hyphal abnormalities, cell wall structural changes, and reduced virulence in murine invasive aspergillosis. Finally, we provide evidence that proper RasA localization is independent of the Ras palmitoyltransferase homolog, encoded by erfB, but requires the palmitoyltransferase complex subunit, encoded by erfD. Our results demonstrate that plasma membrane-associated RasA is critical for polarized morphogenesis, cell wall stability, and virulence in A. fumigatus.

Regulation of expression, activity and localization of fungal chitin synthases.

The fungal cell wall represents an attractive target for pharmacologic inhibition, as many of the components are fungal-specific. Though targeted inhibition of ß-glucan synthesis is effective treatment for certain fungal infections, the ability of the cell wall to dynamically compensate via the cell wall integrity pathway may limit overall efficacy. To date, chitin synthesis inhibitors have not been successfully deployed in the clinical setting. Fungal chitin synthesis is a complex and highly regulated process. Regulation of chitin synthesis occurs on multiple levels, thus targeting of these regulatory pathways may represent an exciting alternative approach. A variety of signaling pathways have been implicated in chitin synthase regulation, at both transcriptional and post-transcriptional levels. Recent research suggests that localization of chitin synthases likely represents a major regulatory mechanism. However, much of the regulatory machinery is not necessarily shared among different chitin synthases. Thus, an in-depth understanding of the precise roles of each protein in cell wall maintenance and repair will be essential to identifying the most likely therapeutic targets.

Localization and activity of the calcineurin catalytic and regulatory subunit complex at the septum is essential for hyphal elongation and proper septation in Aspergillus fumigatus.

Calcineurin, a heterodimer composed of the catalytic (CnaA) and regulatory (CnaB) subunits, plays key roles in growth, virulence and stress responses of fungi. To investigate the contribution of CnaA and CnaB to hyphal growth and septation, ΔcnaB and ΔcnaAΔcnaB strains of Aspergillus fumigatus were constructed. CnaA colocalizes to the contractile actin ring early during septation and remains at the centre of the mature septum. While CnaB's septal localization is CnaA-dependent, CnaA's septal localization is CnaB-independent, but CnaB is required for CnaA's function at the septum. Catalytic null mutations in CnaA caused stunted growth despite septal localization of the calcineurin complex, indicating the requirement of calcineurin activity at the septum. Compared to the ΔcnaA and ΔcnaB strains, the ΔcnaAΔcnaB strain displayed more defective growth and aberrant septation. While three Ca(2+) -binding motifs in CnaB were sufficient for its association with CnaA at the septum, the amino-terminal arginine-rich domains (16-RRRR-19 and 44-RLRKR-48) are dispensable for septal localization, yet required for complete functionality. Mutation of the 51-KLDK-54 motif in CnaB causes its mislocalization from the septum to the nucleus, suggesting it is a nuclear export signal sequence. These findings confirm a cooperative role for the calcineurin complex in regulating hyphal growth and septation.

The chitin synthase genes chsA and chsC are not required for cell wall stress responses in the human pathogen Aspergillus fumigatus.

Invasive aspergillosis is a leading cause of mortality in immunocompromised patients. The fungal cell wall is an attractive antifungal target, but it is dynamic and responsive to external stressors. The existence of multiple chitin synthases within Aspergilli is thought to reflect specialized functions in cell wall damage responses that facilitate continued growth and viability. We previously reported increased transcription of Aspergillus fumigatus chitin synthases chsA and chsC following echinocandin treatment, suggesting important roles for these chitin synthases in cell wall compensation. As only partial disruptions have been made of these genes, we generated deletion mutants of chsA and chsC singly (ΔchsA and ΔchsC) and doubly (ΔchsA ΔchsC). The ΔchsA ΔchsC strain displayed reduced total chitin synthase activity. Interestingly, deletion of these chitin synthase genes did not affect levels of chitin or ß-1,3-glucan.The ΔchsA, ΔchsC and ΔchsA ΔchsC strains produced wild-type echinocandin-mediated chitin increases, consistent with unaltered cell wall compensation. Furthermore, transcript levels of the remaining chitin synthase genes were unchanged in the mutant strains. Taken together, these results indicate that chsA and chsC do not play a direct role in the cell wall stress response. Our findings support the existence of complex post-transcriptional regulatory mechanisms controlling chitin biosynthetic machinery in response to cell wall damage.

Regulatable Ras activity is critical for proper establishment and maintenance of polarity in Aspergillus fumigatus.

Here we show that expression of a constitutively activated RasA allele, as the sole source of Ras activity, revealed novel Ras-induced phenotypes, including excessive vacuolar expansion and spontaneous lysis of hyphal compartments. These findings highlight the requirement for balanced Ras activity in the establishment and maintenance of polarized growth in filamentous fungi.

Differential localization patterns of septins during growth of the human fungal pathogen Aspergillus fumigatus reveal novel functions.

Septins, a conserved family of GTPases, are heteropolymeric filament-forming proteins that associate with the cell membrane and cytoskeleton and serve essential functions in cell division and morphogenesis. Their roles in fungal cell wall chitin deposition, septation, cytokinesis, and sporulation have been well established and they have recently been implicated in tissue invasion and virulence in Candida albicans. Septins have never been investigated in the human pathogenic fungus, Aspergillus fumigatus, which is a leading cause of death in immunocompromised patients. Here we localize all the five septins (AspA-E) from A. fumigatus for the first time, and show that each of the five septins exhibit varied patterns of distribution. Interestingly AspE, which is unique to filamentous fungi, and AspD, belonging to the CDC10 class of septins, localized prominently to tubular structures which were dependent on actin and microtubule networks. Localization of AspD and AspE has never been reported in filamentous fungi. Taken together these results suggest that septins in A. fumigatus might have unique functions in morphogenesis and pathogenicity.

Newer combination antifungal therapies for invasive aspergillosis.

Optimal therapy for invasive aspergillosis is unknown, and many clinicians have attempted to utilize a combination antifungal approach to improve outcomes. However, while numerous in vitro studies, animal models, and clinical reports suggest the possibility that combination antifungal therapy might offer improved results, there is no definitive accepted strategy. The currently available antifungals used in various combination approaches have not demonstrated clear improvement over monotherapy. The current classes of drugs targeting the cell wall and cell membrane may need adjunctive agents focused on separate cellular pathways, such as cell stress response or cellular signaling, to maximize efficacy. The calcineurin and the Hsp90 pathways are two such untouched arenas in which targeted manipulation may lead to great advances against aspergillosis.

Transcriptional regulation of chitin synthases by calcineurin controls paradoxical growth of Aspergillus fumigatus in response to caspofungin.

Attenuated activity of echinocandin antifungals at high concentrations, known as the "paradoxical effect," is a well-established phenomenon in Candida albicans and Aspergillus fumigatus. In the yeast C. albicans, upregulation of chitin biosynthesis via the protein kinase C (PKC), high-osmolarity glycerol response (HOG), and Ca(2+)/calcineurin signaling pathways is an important cell wall stress response that permits growth in the presence of high concentrations of echinocandins. However, nothing is known of the molecular mechanisms regulating the mold A. fumigatus and its paradoxical response to echinocandins. Here, we show that the laboratory strain of A. fumigatus and five of seven clinical A. fumigatus isolates tested display various magnitudes of paradoxical growth in response to caspofungin. Interestingly, none of the eight strains showed paradoxical growth in the presence of micafungin or anidulafungin. Treatment of the DeltacnaA and DeltacrzA strains, harboring gene deletions of the calcineurin A subunit and the calcineurin-dependent transcription factor, respectively, with high concentrations of caspofungin revealed that the A. fumigatus paradoxical effect is calcineurin pathway dependent. Exploring a molecular role for CnaA in the compensatory chitin biosynthetic response, we found that caspofungin treatment resulted in increased chitin synthase gene expression, leading to a calcineurin-dependent increase in chitin synthase activity. Taken together, our data suggest a mechanistic role for A. fumigatus calcineurin signaling in the chitin biosynthetic response observed during paradoxical growth in the presence of high-dose caspofungin treatment.

The Aspergillus fumigatus P-type Golgi apparatus Ca2+/Mn2+ ATPase PmrA is involved in cation homeostasis and cell wall integrity but is not essential for pathogenesis.

The Aspergillus fumigatus DeltapmrA (Golgi apparatus Ca(2+)/Mn(2+) P-type ATPase) strain has osmotically suppressible basal growth defects and cationic tolerance associated with increased expression of calcineurin pathway genes. Despite increased beta-glucan and chitin content, it is hypersensitive to cell wall inhibitors but remains virulent, suggesting a role for PmrA in cation homeostasis and cell wall integrity.