Maria Adelaida Gomez

The Leishmania Surface Protease GP63 Cleaves Multiple Intracellular Proteins and Actively Participates in p38 Mitogen-activated Protein Kinase Inactivation

The Leishmania parasite is a widespread disease threat in tropical areas, causing symptoms ranging from skin lesions to death. Leishmania parasites typically invade macrophages but are also capable of infecting fibroblasts, which may serve as a reservoir for recurrent infection. Invasion by intracellular pathogens often involves exploitation of the host cell cytoskeletal and signaling machinery. Here we have observed a dramatic rearrangement of the actin cytoskeleton and marked modifications in the profile of protein tyrosine phosphorylation in fibroblasts infected with Leishmania major. Correspondingly, exposure to L. major resulted in degradation of the phosphorylated adaptor protein p130Cas and the protein-tyrosine phosphatase-PEST. Cellular and in vitro assays using pharmacological protease inhibitors, recombinant enzyme, and genetically modified strains of L. major identified the parasite protease GP63 as the principal catalyst of proteolysis during infection. A number of additional signaling proteins were screened for degradation during L. major infection as follows: a small subset was cleaved, including cortactin, T-cell protein-tyrosine phosphatase, and caspase-3, but the majority remained unaffected. Protein degradation occurred in cells incubated with Leishmania extracts in the absence of intact parasites, suggesting a mechanism permitting transfer of functional GP63 into the intracellular space. Finally, we evaluated the impact of Leishmania on MAPK signaling; unlike p44/42 and JNK, p38 was inactivated upon infection in a GP63- and protein degradation-dependent manner, which likely involves cleavage of the upstream adaptor TAB1. Our results establish that GP63 plays a central role in a number of hostcell molecular events that likely contribute to the infectivity of Leishmania.

Host cell signalling and leishmania mechanisms of evasion.

Leishmania parasites are able to secure their survival and propagation within their host by altering signalling pathways involved in the ability of macrophages to kill pathogens or to engage adaptive immune system. An important step in this immune evasion process is the activation of host protein tyrosine phosphatase SHP-1 by Leishmania. SHP-1 has been shown to directly inactivate JAK2 and Erk1/2 and to play a role in the negative regulation of several transcription factors involved in macrophage activation. These signalling alterations contribute to the inactivation of critical macrophage functions (e.g., Nitric oxide, IL-12, and TNF-α). Additionally, to interfere with IFN-γ receptor signalling, Leishmania also alters several LPS-mediated responses. Recent findings from our laboratory revealed a pivotal role for SHP-1 in the inhibition of TLR-induced macrophage activation through binding to and inactivating IL-1-receptor-associated kinase 1 (IRAK-1). Furthermore, we identified the binding site as an evolutionarily conserved ITIM-like motif, which we named kinase tyrosine-based inhibitory motif (KTIM). Collectively, a better understanding of the evasion mechanisms utilized by Leishmania parasite could help to develop more efficient antileishmanial therapies in the near future.

Discovery of factors linked to antimony resistance in Leishmania panamensis through differential proteome analysis

The rate of treatment failure to antileishmanial chemotherapy in Latin America is up to 64%. Parasite drug resistance contributes to an unknown proportion of treatment failures. Identification of clinically relevant molecular mechanisms responsible for parasite drug resistance is critical to the conservation of available drugs and to the discovery of novel targets to reverse the resistant phenotype. We conducted comparative proteomic-based analysis of Leishmania (Viannia) panamensis lines selected in vitro for resistance to trivalent antimony (Sb(III)) to identify factors associated with antimony resistance. Using 2-dimensional gel electrophoresis, two distinct sub-proteomes (soluble in NP-40/urea and Triton X-114, respectively) of promastigotes of WT and Sb(III)-resistant lines were generated. Overall, 9 differentially expressed putative Sb-resistance factors were detected and identified by mass spectrometry. These constituted two major groups: (a) proteins involved in general stress responses and (b) proteins with highly specific metabolic and transport functions, potentially directly contributing to the Sb-resistance mechanism. Notably, the sulfur amino acid-metabolizing enzymes S-adenosylmethionine synthetase (SAMS) and S-adenosylhomocysteine hydrolase (SAHH) were over-expressed in Sb(III)-resistant lines and Sb(III)-resistant clinical isolates. These enzymes play a central role in the upstream synthesis of precursors of trypanothione, a key molecule involved in Sb-resistance in Leishmania parasites, and suggest involvement of epigenetic regulation in response to drug exposure. These data re-enforce the importance of thiol metabolism in Leishmania Sb resistance, reveal previously unrecognized steps in the mechanism(s) of Sb tolerance, and suggest a cross-talk between drug resistance, metabolism and virulence.

NRAMP-1 expression modulates protein tyrosine phosphatase activity in macrophages: Impact on host cell signaling and functions

NRAMP-1 (natural resistance-associated macrophage protein-1) has been associated with innate resistance to unrelated intracellular pathogen infections, up-regulation of proinflammatory phagocyte functions, and susceptibility to autoimmune diseases. It is still unclear how the divalent cation transport function of NRAMP-1 accounts for the associated pleiotropic effects. In this study, we evaluated the impact of murine macrophage NRAMP-1 expression on the activity of protein-tyrosine phosphatases (PTPs) as an upstream event contributing to the NRAMP-1 regulation of signal transduction and control of effector macrophage functions. Functional expression of NRAMP-1 results in lower macrophage PTP activity and increased protein phosphorylation. Decreased PTP activity is not a result of changes in protein expression but rather a reversible regulatory mechanism involving the interaction with NRAMP-1 metal substrates. In the context of intracellular infections, NRAMP-1 expression prevents full macrophage PTP induction by Leishmania infection, correlating with higher nitric oxide production and lower parasite survival. We suggest that NRAMP-1 divalent cation transport leads to transient inhibition of PTPs via direct PTP-metal interaction and/or by reactive oxygen species-dependent PTP oxidation, consequently promoting positive signal transduction, as a backbone for the induction of proinflammatory phagocyte functions.

Treatment Failure and Miltefosine Susceptibility in Dermal Leishmaniasis Caused by Leishmania Subgenus Viannia Species

ABSTRACT Treatment failure and parasite drug susceptibility in dermal leishmaniasis caused by Leishmania (Viannia) species are poorly understood. Prospective evaluation of drug susceptibility of strains isolated from individual patients before drug exposure and at clinical failure allows intrinsic and acquired differences in susceptibility to be discerned and analyzed. To determine whether intrinsic susceptibility or loss of susceptibility to miltefosine contributed to treatment failure, we evaluated the miltefosine susceptibility of intracellular amastigotes and promastigotes of six Leishmania (Viannia) braziliensis and six Leishmania (Viannia) panamensis strains isolated sequentially, at diagnosis and treatment failure, from two children and four adults ≥55 years old with concurrent conditions. Four patients presented only cutaneous lesions, one had mucosal disease, and one had disseminated mucocutaneous disease. Expression of the Leishmania drug transporter genes abca2, abca3, abcc2, abcc3, abcg4, abcg6, and LbMT was evaluated by quantitative reverse transcription-PCR (qRT-PCR). Intracellular amastigotes (median 50% effective concentration [EC50], 10.7 μmol/liter) were more susceptible to miltefosine than promastigotes (median EC50, 55.3 μmol/liter) (P < 0.0001). Loss of susceptibility at failure, demonstrated by a miltefosine EC50 of >32 μmol/liter (the upper limit of intracellular amastigote assay), occurred in L. panamensis infection in a child and in L. braziliensis infection in an adult and was accompanied by decreased expression of the miltefosine transporter LbMT (LbMT/β-tubulin, 0.42- to 0.26-fold [P = 0.039] and 0.70- to 0.57-fold [P = 0.009], respectively). LbMT gene polymorphisms were not associated with susceptibility phenotype. Leishmania ABCA3 transporter expression was inversely correlated with miltefosine susceptibility (r = −0.605; P = 0.037). Loss of susceptibility is one of multiple factors involved in failure of miltefosine treatment in dermal leishmaniasis.

Sensitive diagnosis of cutaneous leishmaniasis by lesion swab sampling coupled to qPCR

Variation in clinical accuracy of molecular diagnostic methods for cutaneous leishmaniasis (CL) is commonly observed depending on the sample source, the method of DNA recovery and the molecular test. Few attempts have been made to compare these variables. Two swab and aspirate samples from lesions of patients with suspected CL (n = 105) were evaluated alongside standard diagnosis by microscopic detection of amastigotes or culture of parasites from lesion material. Three DNA extraction methods were compared: Qiagen on swab and aspirate specimens, Isohelix on swabs and Boil/Spin of lesion aspirates. Recovery of Leishmania DNA was evaluated for each sample type by real-time polymerase chain reaction detection of parasitic 18S rDNA, and the diagnostic accuracy of the molecular method determined. Swab sampling combined with Qiagen DNA extraction was the most efficient recovery method for Leishmania DNA, and was the most sensitive (98%; 95% CI: 91-100%) and specific (84%; 95% CI: 64-95%) approach. Aspirated material was less sensitive at 80% (95% CI: 70-88%) and 61% (95% CI: 50-72%) when coupled to Qiagen or Boil-Spin DNA extraction, respectively. Swab sampling of lesions was painless, simple to perform and coupled with standardized DNA extraction enhances the feasibility of molecular diagnosis of CL.

Miltefosine and Antimonial Drug Susceptibility of Leishmania Viannia Species and Populations in Regions of High Transmission in Colombia

Background Pentavalent antimonials have been the first line treatment for dermal leishmaniasis in Colombia for over 30 years. Miltefosine is administered as second line treatment since 2005. The susceptibility of circulating populations of Leishmania to these drugs is unknown despite clinical evidence supporting the emergence of resistance. Methodology/Principal Findings In vitro susceptibility was determined for intracellular amastigotes of 245 clinical strains of the most prevalent Leishmania Viannia species in Colombia to miltefosine (HePC) and/or meglumine antimoniate (SbV); 163, (80%) were evaluated for both drugs. Additionally, susceptibility to SbV was examined in two cohorts of 85 L. V. panamensis strains isolated between 1980–1989 and 2000–2009 in the municipality of Tumaco. Susceptibility to each drug differed among strains of the same species and between species. Whereas 68% of L. V. braziliensis strains presented in vitro resistance to HePC, 69% were sensitive to SbV. Resistance to HePC and SbV occurred respectively, in 20% y 21% of L. panamensis strains. Only 3% of L. V. guyanensis were resistant to HePC, and none to SbV. Drug susceptibility differed between geographic regions and time periods. Subpopulations having disparate susceptibility to SbV were discerned among L. V. panamensis strains isolated during 1980–1990 in Tumaco where resistant strains belonged to zymodeme 2.3, and sensitive strains to zymodeme 2.2. Conclusions/Significance Large scale evaluation of clinical strains of Leishmania Viannia species demonstrated species, population, geographic, and epidemiologic differences in susceptibility to meglumine antimoniate and miltefosine, and provided baseline information for monitoring susceptibility to these drugs. Sensitive and resistant clinical strains within each species, and zymodeme as a proxy marker of antimony susceptibility for L. V. panamensis, will be useful in deciphering factors involved in susceptibility and the distribution of sensitive and resistant populations.

The shape of the HIV/AIDS epidemic in Puerto Rico

This study presents information on AIDS patients in Puerto Rico, including their general sociodemographic profile, some risk-related parameters, characteristics of vulnerable groups, and elements of the clinical spectrum of the disease. Data were analyzed from the Puerto Rico AIDS Surveillance Program and available studies about the HIV/AIDS epidemic in Puerto Rico. A total of 23,089 AIDS cases was reported to the Puerto Rico AIDS Surveillance Program from January 1981 through February 1999. The HIV/AIDS epidemic has affected mostly males and females between the ages of 30 and 49, though cases have also been reported for other age groups. The cumulative proportion of persons with AIDS who are women has increased tremendously, from 11.4% for the 1981-1986 period to 21.6% for the entire 1981-1999 period. In Puerto Rico the category of injecting drug users (IDUs) accounts for the majority of the AIDS cases (52%), followed by heterosexual contact (22%), and men who have sex with men (17%). The three main diagnoses for AIDS on the island are wasting syndrome (30.7%); esophageal, bronchial, and lung candidiasis (29.4%); and Pneumocystis carinii pneumonia (26.8%). According to 1994 vital statistics for Puerto Rico, AIDS was the fourth-leading cause of death. The overall reported AIDS mortality rate was 42.0 per 100,000 persons, with the rate for males, 67.8, much higher than it was for females, 17.4. AIDS is the first cause of death among persons between 30 and 39 years old. Intense efforts are needed to better understand the epidemic in Puerto Rico and its biology, social and family impacts, and financial costs.

Leishmania (Viannia) Infection in the Domestic Dog in Chaparral, Colombia

Peridomestic transmission of American cutaneous leishmaniasis is increasingly reported and dogs may be a reservoir of Leishmania (Viannia) in this setting. We investigated the prevalence of infection in dogs in Chaparral County, Colombia, the focus of an epidemic of human cutaneous leishmaniasis caused by Leishmania (Viannia) guyanensis. Two (0.72%) of 279 dogs had lesions typical of cutaneous leishmaniasis that were biopsy positive by kinetoplast DNA polymerase chain reaction-Southern blotting. Seroprevalence was 2.2% (6 of 279) by enzyme-linked immunosorbent assay. Buffy coat and ear skin biopsy specimens were positive by polymerase chain reaction-Southern blotting in 7.3% (10 of 137) and 11.4% (12 of 105) of dogs, respectively. Overall 20% of dogs (21 of 105) showed positive results for one or more tests. Amplification and sequencing of the Leishmania 7SL RNA gene identified L. guyanensis in one dog and L. braziliensis in two dogs. No association was identified between the risk factors evaluated and canine infection. Dogs may contribute to transmission but their role in this focus appears to be limited.

Leishmania panamensis infection and antimonial drugs modulate expression of macrophage drug transporters and metabolizing enzymes: impact on intracellular parasite survival

OBJECTIVESnTreatment failure is multifactorial. Despite the importance of host cell drug transporters and metabolizing enzymes in the accumulation, distribution and metabolism of drugs targeting intracellular pathogens, their impact on the efficacy of antileishmanials is unknown. We examined the contribution of pharmacologically relevant determinants in human macrophages in the antimony-mediated killing of intracellular Leishmania panamensis and its relationship with the outcome of treatment with meglumine antimoniate.nnnMETHODSnPatients with cutaneous leishmaniasis who failed (n = 8) or responded (n =8) to treatment were recruited. Gene expression profiling of pharmacological determinants in primary macrophages was evaluated by quantitative RT-PCR and correlated to the drug-mediated intracellular parasite killing. Functional validation was conducted through short hairpin RNA gene knockdown.nnnRESULTSnSurvival of L. panamensis after exposure to antimonials was significantly higher in macrophages from patients who failed treatment. Sixteen macrophage drug-response genes were modulated by infection and exposure to meglumine antimoniate. Correlation analyses of gene expression and intracellular parasite survival revealed the involvement of host cell metallothionein-2A and ABCB6 in the survival of Leishmania during exposure to antimonials. ABCB6 was functionally validated as a transporter of antimonial compounds localized in both the cell and phagolysosomal membranes of macrophages, revealing a novel mechanism of host cell-mediated regulation of intracellular drug exposure and parasite survival within phagocytes.nnnCONCLUSIONSnThese results provide insight into host cell mechanisms regulating the intracellular exposure of Leishmania to antimonials and variations among individuals that impact parasite survival. Understanding of host cell determinants of intracellular pharmacokinetics/pharmacodynamics opens new avenues to improved drug efficacy for intracellular pathogens.