Antonella Valerio

Mutations in the Dihydropteroate Synthase Gene of Human-Derived Pneumocystis carinii Isolates from Italy Are Infrequent but Correlate with Prior Sulfa Prophylaxis

Mutations in the human-derived Pneumocystis carinii dihydropteroate synthase (DHPS) gene have been reported with increasing frequency and have been linked to prior sulfa prophylaxis and possible emergence of sulfa resistance. This study was done to examine the prevalence and clinical significance of P. carinii DHPS mutations in Italian patients. A previously described single-strand conformation polymorphism technique was used to identify P. carinii DHPS mutations in 107 patients with acquired immunodeficiency syndrome. Overall prevalence (8%) was low compared with that in other reports. Mutations were observed in 19% (6/31) of patients exposed to sulfa prophylaxis, compared with 4% (3/76) of patients not exposed to sulfa prophylaxis (P=.017). No significant association was observed between the presence of DHPS mutations and mortality, CD4 cell count, or demographic factors. The study confirms the association between DHPS mutations and prior sulfa prophylaxis and shows that the prevalence of DHPS mutations in an Italian patient population is lower than that in other populations.

Genotyping of Pneumocystis jiroveci pneumonia in Italian AIDS patients. Clinical outcome is influenced by dihydropteroate synthase and not by internal transcribed spacer genotype.

Background:Two Pneumocystis jiroveci independent genomic regions, internal transcribed spacer (ITS) 1 and ITS2, and dihydropteroate synthase (DHPS) gene have been used for typing a cohort of HIV-infected Italian patients with P. jiroveci pneumonia (PcP). Methods:Bronchoalveolar lavage samples isolated from 207 HIV-infected adults were ITS and DHPS genotyped by DNA sequencing and by restriction fragment length polymorphism analysis, respectively. Mutant DHPS samples were cloned and ITS typed. Data on severity, treatment, and outcome of PcP were obtained by chart review. Results:High diversity with 46 different ITS genotypes was observed. At the DHPS locus, 9.1% of samples analyzed were found to be mutated. A correlation was observed between DHPS mutants and greater severity of PcP, as defined by higher lactate dehydrogenase (P = 0.015) and need for intubation (P = 0.002), and worse outcomes, as defined by failure of sulfa treatment (P = 0.04), death, and/or relapse of PcP (P = 0.008). There was a significant difference in ITS genotype patterns between DHPS wild-type and mutants (P = 0.028). Conclusions:The present data suggest the absence of a correlation between P. jiroveci ITS types and specific clinical characteristics. DHPS mutations correlate with possible failure of anti-P. jiroveci sulfa therapy, and a trend of association is shown between DHPS mutations and some clinical PcP features.

DHPS‐Mutated Isolates of Pneumocystis jirovecii from HIV‐Infected Individuals: Analysis of Related ITS Genotypes

PNEUMONIA caused by Pneumocystis jirovecii remains the most common opportunistic pneumonia in AIDS and other immunocompromised patients (Morris et al. 2004). Most drugs used for prevention and treatment of Pneumocystis pneumonia (PcP) target enzymes involved in the biosynthesis of folic acid; sulfa drugs, in particular sulfamethoxazole (SMX), inhibit the dihydropteroate synthase (DHPS). Emergence of sulfa drug-resistant P. jirovecii has been suggested to be due to exposure to sulfa drugs and leading to DHPS mutations. Two point mutations corresponding to amino acid positions 55 and 57 of the P. jirovecii DHPS protein are the genetic markers for possible sulphonamide resistance that have been reported with increasing frequency (Kazanjian et al. 2004, 2000; Lane et al. 1997; Ma et al. 1999). In this study, the DHPS and the internal transcribed spacers (ITS) loci of P. jirovecii isolates from HIV-infected patients were typed to investigate the possible association between specific ITS and DHPS types and what that might mean in the epidemiology of PcP. MATERIALS AND METHODS

Pneumocystis carinii ITS Typing: Doubtful Evidence of Genotype-Related Virulence

Pnewnocystis carinii is an oppurtunistic pulmonary pathogen particularly associated with HIV-infected patients. Controversial results have been reparted on the possible occurrence of genotyperelated virulence [l-21 based on Internal Transcribed Spacers (ITS) typing. Wide ITS variability has been identified. and the locus is described as a single copy gene, thus allowing accurate description of genotypes during P. carinii pneumonia (PCP) episodes [3, 41. Based on paired cliical and molecular analysis of lTS sequences, we examined if there is a correlation between these two parameters among P. carinii isolated fkom patients in Italy.

The Non‐Peptidic HIV Protease Inhibitor Tipranavir and Two Synthetic Peptidomimetics (TS98 and TS102) Modulate Pneumocystis carinii Growth and Proteasome Activity of HEL299 Cell Line

THE clinical efficacy of highly active antiretroviral therapy (HAART) containing HIV protease inhibitors against opportunistic infections is now well known (Sepkowitz 1998). Widely used HIV protease inhibitors (including Saquinavir, Ritonavir, Indinavir, Nelfinavir, Amprenavir, Lopinavir, and Atazanavir) are compounds with structures that mimic the local topography around an amide bond. They are low MW peptidomimetics (MWo1,000 Da) containing transition-state isostere (hydroxyethylamine and hydroxyethylene) that match the peptide backbone atom-for-atom and form important contacts with the HIV protease binding-site (Ripka and Rich 1998). We previously demonstrated that the majority of available HIV protease inhibitors partially and non-specifically reduced rat-derived Pneumocystis carinii growth in vitro when tested at clinically administered concentrations (Atzori et al. 2000, 2001; Mazza 2003). In this study we evaluated the anti-Pneumocystis activity of three other compounds, Tipranavir, TS98, and TS102. TS98 and TS102 are small peptidomimetics that are structurally and functionally correlated with clinical HIV protease inhibitors. TS98 and TS102 were designed by taking into account the binding site of HIV-1 and Candida albicans (Sap2) proteases (Tossi et al. 2003). Tipranavir is the first compound of a new class of non-peptidic HIV protease inhibitors based on novel templates which, though appearing unrelated to the original peptides, contain the necessary groups positioned on a non-peptidic scaffold that serves as a topographical mimetic. This kind of molecules represents an ideal peptidomimetic in that their non-peptidic nature makes them recalcitrant to proteolytic degradation and hence have increased bioavailability (Ripka and Rich 1998). Because of the lack of a continuous in vitro culture system for P. carinii, we assayed Tipranavir, TS98, and TS102 in a monoxenic culture system containing P. carinii isolated from infected rat lungs and human embryonic lung cells (HEL299) feeder cells. To understand if the effects observed on P. carinii proliferation in vitro were related to modulation of feeder cell proteasomes, we performed examined the effect of the three compounds on intact HEL299 cells and the 20S proteasome isolated from them (Groettrup et al. 1995). The 26S proteasome is a multifunctional proteolytic complex of approximately 2,000 kDa consisting of two subcomplexes. These include the 20S proteasome and the 19S particle that serve catalytic and regulatory functions, respectively. The 26S proteasome is able to degrade proteins with almost complete specificity for ubiquitin-conjugated proteins, and its catalytic activity depends on ATP. In vivo, the 20S proteasome is likely to exist as a single particle or in combination with the 19S regulator. It can also combine with another particle, 11S. 11S plays a regulatory role in producing the hybrid 19S–20S–11S particle, which requires ATP for its assembly and retains the ability to degrade ubiquitinated conjugates. Proteasomes purified from different tissues and species are approximately 700 kDa in size and are comprised of four staked rings, each containing multiple subunits. This particle designated as the 20S proteasome retains peptidase activity without requiring ATP and is unable by itself to degrade proteins combined with ubiquitin molecules (Piccinini, Mostert, and Rinaudo 2003).

Detection of HIV protease inhibitors in alveolar epithelial lining fluid: relevance for modulation of pneumocystis infection in the course of HAART.

RECENT studies indicate that there is an increased survival rate among HIV-infected patients with severe Pneumocystis jirovecii pneumonia when treated with highly active antiretroviral therapy (HAART; Morris et al. 2003). Explanation for the survival benefit with HAART include decreased HIV fitness, attenuated rise in HIV load, or anti-Pneumocystis activities of HIV protease inhibitors. It has been demonstrated that several HIV protease inhibitors exert in vitro anti-Pneumocystis effects at concentrations achievable in vivo during HAART (Atzori et al. 2000, 2001, 2002; Atzori and Cargnel 2002; Cassone et al. 1999; Derouin and Santillana-Hayat 2002; Korting et al. 1999). The clinical relevance of this observation remains to be investigated. A reliable method to measure the concentration of antiPneumocystis drugs such as pentamidine in lung epithelial lining fluid (ELF; Conte et al. 1999) has been successfully applied to detect Lopinavir in an HIV patient (Atzori et al. 2003). Lopinavir is well known for its high achievable blood concentration during clinical use, but it is detectable in alveolar epithelial lining fluid only by co-administration of small amount of Ritonavir (RTV); this process is commonly referred to as ‘‘boosting.’’ In this study we evaluated the ability to detect in alveolar lining fluid other HIV protease inhibitors including Nelfinavir, Saquinavir, and Tipranavir with Ritonavir boosting during administration of HAART.

HIV Protease Inhibitors and Pneumocystis

neumacystis is a major cause of pneumonia in patients with P AIDS and in other immunocompromised patients. Since the introduction of HIV protease inhibitors (PIS), dramatic declines in all opportunistic infections, including Pneumocystis pneumonia (PcP), has been noticed among AIDS patients [ 1 11. A recent study reported improved survival with highly active antiretroviral therapy (HAART) in HIV-infected patients with severe PcP [lo]. Explanation for possible survival benefits from HAART includes reduction in viral load and alteration of predominant strain to a less fit variant allowing more CD4 cells to survive. However, protease inhibitors could have other effects including effects on organisms other than HIV. We first demonstrated by zymograms the presence of seven cysteine-serine type proteases (molecular weight range: 40-98 kDa) in rat-derived P. curinii trophozoites by using gelatin SDS-PAGE (Atzori, C., Means, I., Bartlett, M. S., Smith, J. W. & Queener, S. 1991. American Society of Microbiology (abstract), B98:42). Other groups then showed a P. carinii protease multi-gene family with homology to subtilisin-like serine proteases [12]. The presence of aspartyl proteases in P. carinii trophozoite zymograms and anti-Pneumocystis dose-dependent activity of Pepstatin A (progenitor of acid proteases) in a well established in vitro model were then described. P. carinii trophozoites from the animal model were inoculated onto human embryonic lung cells (HEL 299, ATCC KCL137) maintained in multiwell plates [9]. Confluent monolayers were inoculated with P. curinii and incubated in the presence of increasing concentrations of several HIV PIS. Ritonavir (RTV), Saquinavir (SQV), Indinavir (IDV), Nelfinavir (NFV), Amprenavir (APV) and Lopinavir (LPV) inhibited the growth of P. carinii at concentrations clinically achievable in vivo as summarized in Table I [3,4]. Among anti-Pneumocystis PIS, the dose-dependent effect was particularly evident for APV, a sulphamide-like drug. The newer HIV PI Atazanavir, was also tested against P. carinii. This drug surprisingly increased the number of trophozoites in a dose-dependent fashion in repeated experiments. These data indicate that not all PIS have anti-Pneumocystis activity, but that specific features of the drug can result in a completely different effect on P. curinii growth in vitro. Compared to other HIV PIS, the effect of Atazanavir may be explained by its biochemical features, namely the chemical symmetry at C-2.

No Correlation Between ITS Genotypes and DHPS Mutations

eumocystis is an opportunistic pulmonary pathogen particularly associated with HIV-infected patients. Several studies have demonstrated a significant association between the use of sulfa drugs for Pneumcystis pneumonia (PcP) prophylaxis and the presence of DHPS (dihydropteroate synthase) gene point mutations, mainly at position 55 and 57 in the enzyme active site [1,2]. Pneumocystis DHPS mutations have been reported to be increasing in several countries and this may be an emerging public health problem. To understand how these mutations arose we investigated whether DHPS mutations were associated with specific P. jirovecii strains by also sequencing the Internally Transcribed Spacers (ITS) region of DHPS mutants.

PCP Occurring in HIV Patients Under HAART: Assessment of Specific Immunity Against P. carinii

The beneficial effect of highly active anti-retroviral therapy (HAART) in reducing opportunistic infections associated with AIDS is currently attributed to viral suppression and T-cell reconstitution. Observational studies suggested that P. carinii prophylaxis can be safely discontinued when CD4>200 [2]. We recently observed hvo P. carinii pneumonia (PCP) with CD4 >250 under stable HAART. We decided to study lymph-proliferative in vitro response to non-specific and P. carinii-specific antigens (PcAg) in index cases and controls.

Detection of a proteasome in Pneumocystis carinii and its modulation by specific proteasome inhibitors.

PNEUMOCYSTIS is a major cause of pneumonia (PcP) and morbidity in patients with an impaired immune response (Stringer and Walzer 1996). Better survival has been observed among HIV-infected patients with severe PcP receiving highly active antiretroviral therapy (HAART) (Sepkowitz 1998; Morris et al. 2003). The anti-opportunistic infection benefits of HAART could be explained by reduced viral fitness and hence an attenuated rise in viral load during PcP. However, the drop in PcP episodes and other opportunistic infections was also observed in HIV patients even when HAART failed to affect viral load or before restoration of immune responses (Atzori et al. 2003), suggesting that alternative mechanisms were playing a role. After HIV protease inhibitors were introduced into HAART regimens a dramatic decline in the incidence of opportunistic infections has been observed among AIDS patients (Sepkowitz 1998). We previously tested protease inhibitors on rat-derived P. carinii using a monoxenic primary culture system containing HEL 299 cells adhered to microcarrier beads. We observed partial, non-specific growth inhibition when testing most protease inhibitors available to us (Atzori et al. 2000, 2001). In those studies, protease inhibitor concentrations were similar to those used clinically. These observations led us to investigate the role of proteasomes in PcP therapy. We hypothesized that the reduction of P. carinii proliferation in response to protease inhibitors occurred in part by negative modulation of proteasome function. Proteasomes are multi-unit neutral proteases distributed in the cytoplasmic and nuclear compartments of cells that catalyze ubiquitin/ATP-dependent protein degradation. This system is involved in (a) regular turnover of intracellular proteins, (b) rapid degradation of abnormal proteins as well as critical regulatory proteins, (c) modulation of cell surface receptors and ion channels, and (d) degradation of antigenic proteins producing peptides that are assembled with MHC-I molecules for exposure on the cell surface (Piccinini et al. 2000). The presence of a proteasome in Pneumocystis is still debated; progress in its characterization has been hampered by the lack of a continuous in vitro culture system. However, because Pneumocystis is a eukaryotic microorganism, it is likely that it possesses proteasomes like other eukaryotes. Our current study focused on detecting the proteasome in P. carinii. We tested the effects of the protease inhibitor Lopinavir on the putative proteasome peptidase activity and the proteasome-specific inhibitors Lactacystin, Epoxomicine, and Bortezomib as well. The effect of Bortezomib on growth of P. carinii in vitro was also examined.