Marta H. Branquinha

Detection of extracellular proteases from microorganisms on agar plates

We present herein an improved assay for detecting the presence of extracellular proteases from microorganisms on agar plates. Using different substrates (gelatin, BSA, hemoglobin) incorporated into the agar and varying the culture medium composition, we were able to detect proteolytic activities from Pseudomonas aeruginosa, Micrococcus luteus and Serratia marcescens as well as the influence that these components displayed in the expression of these enzymes. For all microorganisms tested we found that in agar-BHI or yeast extract medium containing gelatin the sensitivity of proteinase detection was considerably greater than in BSA-agar or hemoglobin-agar. However, when BSA or hemoglobin were added to the culture medium, there was an increase in growth along with a marked reduction in the amount of proteinase production. In the case of M. luteus the incorporation of glycerol in BHI or yeast extract gelatin-agar induced protease liberation. Our results indicate that the technique described here is of value for detecting extracellular proteases directly in the culture medium, by means of a qualitative assay, simple, inexpensive, straight forward method to assess the presence of the proteolytic activity of a given microorganism colony with great freedom in substrate selection.

HIV Aspartyl Peptidase Inhibitors Interfere with Cellular Proliferation, Ultrastructure and Macrophage Infection of Leishmania amazonensis

Background Leishmania is the etiologic agent of leishmanisais, a protozoan disease whose pathogenic events are not well understood. Current therapy is suboptimal due to toxicity of the available therapeutic agents and the emergence of drug resistance. Compounding these problems is the increase in the number of cases of Leishmania-HIV coinfection, due to the overlap between the AIDS epidemic and leishmaniasis. Methodology/Principal Findings In the present report, we have investigated the effect of HIV aspartyl peptidase inhibitors (PIs) on the Leishmania amazonensis proliferation, ultrastructure, interaction with macrophage cells and expression of classical peptidases which are directly involved in the Leishmania pathogenesis. All the HIV PIs impaired parasite growth in a dose-dependent fashion, especially nelfinavir and lopinavir. HIV PIs treatment caused profound changes in the leishmania ultrastructure as shown by transmission electron microscopy, including cytoplasm shrinking, increase in the number of lipid inclusions and some cells presenting the nucleus closely wrapped by endoplasmic reticulum resembling an autophagic process, as well as chromatin condensation which is suggestive of apoptotic death. The hydrolysis of HIV peptidase substrate by L. amazonensis extract was inhibited by pepstatin and HIV PIs, suggesting that an aspartyl peptidase may be the intracellular target of the inhibitors. The treatment with HIV PIs of either the promastigote forms preceding the interaction with macrophage cells or the amastigote forms inside macrophages drastically reduced the association indexes. Despite all these beneficial effects, the HIV PIs induced an increase in the expression of cysteine peptidase b (cpb) and the metallopeptidase gp63, two well-known virulence factors expressed by Leishmania spp. Conclusions/Significance In the face of leishmaniasis/HIV overlap, it is critical to further comprehend the sophisticated interplays among Leishmania, HIV and macrophages. In addition, there are many unresolved questions related to the management of Leishmania-HIV-coinfected patients. For instance, the efficacy of therapy aimed at controlling each pathogen in coinfected individuals remains largely undefined. The results presented herein add new in vitro insight into the wide spectrum efficacy of HIV PIs and suggest that additional studies about the synergistic effects of classical antileishmanial compounds and HIV PIs in macrophages coinfected with Leishmania and HIV-1 should be performed.

Ubiquity of Cysteine- and Metalloproteinase Activities in a Wide Range of Trypanosomatids

We have analysed the proteinase profiles of 11 species from 7 different genera of trypanosomatids by in situ detection of enzyme activities on SDS‐PAGE gels containing co‐polymerized gelatin as substrate, and the use of specific proteinase inhibitors. Our survey indicates that while cysteine‐ and metalloproteinases are distributed ubiquitously among trypanosomatids, there are marked differences between the enzyme profiles from the monogenetic (Crithidia, Herpetomonas, Leptomonas) and digenetic (Trypanosoma, Endotrypanum, Phytomonas, Leishmania) species. The detected metalloproteinase activities, ranging in size from 50–100 kDa, partitioned into the detergent‐phase after Triton X‐114 extraction, while most of cysteine proteinases, of three distinct molecular mass ranges (30–50 kDa, 80–100 kDa and 116–205 kDa), partitioned into the aqueous phase. Thus, within this group of organisms, the metalloproteinase activities seem to be predominantly membrane‐associated proteins. We also show that the plant parasites of the genus Phytomonas exhibit a distinctive cysteine proteinase profile that might be exploited further as a criterion for taxonomy of the genus.

Proteolytic expression in Blastocrithidia culicis : influence of the endosymbiont and similarities with virulence factors of pathogenic trypanosomatids

Blastocrithidia culicis is an insect trypanosomatid that presents bacterial endosymbionts. The cell-associated and secreted proteinases of the endosymbiont-bearing and aposymbiotic strains were compared through the incorporation of proteinaceous substrates into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Few qualitative changes could be detected in the proteolytic zymograms in the 2 strains studied when gelatin, casein, haemoglobin or bovine serum albumin (BSA) were tested. However, the level of proteolytic activities was significantly higher in the aposymbiotic strain. Some of the B. culicis proteins reacted in Western blots with antibodies raised against gp63, a zinc-metalloproteinase, and cruzipain, a cysteinyl-proteinase, which are virulence factors of the human pathogenic trypanosomatids, Leishmania spp. and Trypanosoma cruzi, respectively. The anti-cross-reacting determinant (CRD) antibody recognized 2 polypeptides (50 and 58 kDa) in the spent culture media and in the supernatant from glycosylphosphatidylinositol-phospholipase C (GPI-PLC)-treated cells, suggesting that these proteins are GPI-anchored to the plasma membrane. In addition, the anti-gp63 reacted with the 50 kDa protein. The identification of protein homologues in trypanosomatids with distinct life-cycles may help to determine the importance of proteinases in trypanosomatids.

Miltefosine induces programmed cell death in Leishmania amazonensis promastigotes

In the current study, we evaluated the mechanism of action of miltefosine, which is the first effective and safe oral treatment for visceral leishmaniasis, in Leishmania amazonensis promastigotes. Miltefosine induced a process of programmed cell death, which was determined by the externalization of phosphatidylserine, the incorporation of propidium iodide, cell-cycle arrest at the sub-G0/G1 phase and DNA fragmentation into oligonucleosome-sized fragments. Despite the intrinsic variation that is detected in Leishmania spp, our results indicate that miltefosine causes apoptosis-like death in L. amazonensis promastigote cells using a similar process that is observed in Leishmania donovani.

Arrested growth of Trypanosoma cruzi by the calpain inhibitor MDL28170 and detection of calpain homologues in epimastigote forms

In this paper, we aimed to explore the effects of the calpain inhibitor III (MDL28170) and to detect calpain-like molecules (CALPs) in epimastigote forms of Trypanosoma cruzi isolate Dm28c. MDL28170 at 70 microM promoted a powerful reduction in the growth rate after 48 h. The IC50 value was calculated to be 31.7 microM. This inhibitor promoted an increase in the cellular volume, but not cell lysis, resulting in a trypanostatic effect. T. cruzi CALPs presented a strong cross-reactivity with anti-Drosophila melanogaster calpain and anti-cytoskeleton-associated protein from Trypanosoma brucei antibodies, and labelling was found mainly intracellularly. Furthermore, an 80 kDa reactive protein was detected by Western blotting assays. No significant cross-reactivity was found with anti-human brain calpain antibody. The expression of CALPs was decreased in cells kept for long periods in axenic cultures in comparison to a strain recently isolated from mice, as well as in MDL28170-treated cells, the latter being paralleled by an increased expression of cruzipain. Different levels of CALPs expression were also detected in distinct phylogenetic lineages, like Y strain (lineage TcII), Dm28c (lineage TcI) [corrected] and INPA6147 strain (Z3 zymodeme). These results may contribute for the investigation of the functions of CALPs in trypanosomatids.

Antimicrobial Action of Chelating Agents: Repercussions on the Microorganism Development, Virulence and Pathogenesis

Infections caused by resistant microorganisms often fail to respond to conventional therapy, resulting in prolonged illness, increased treatment costs and greater risk of death. Consequently, the development of novel antimicrobial drugs is becoming more demanding every day since the existing drugs either have too many side-effects or they tend to lose effectiveness due to the selection of resistant strains. In view of these facts, a number of new strategies to obstruct vital biological processes of a microbial cell have emerged; one of these is focused on the use of metal-chelating agents, which are able to selectively disturb the essential metal metabolism of the microorganism by interfering with metal acquisition and bioavailability for crucial reactions. The chelation activity is able to inhibit the biological role of metal-dependent proteins (e.g., metalloproteases and transcription factors), disturbing the microbial cell homeostasis and culminating in the blockage of microbial nutrition, growth and development, cellular differentiation, adhesion to biotic (e.g., extracellular matrix components, cell and/or tissue) and abiotic (e.g., plastic, silicone and acrylic) structures as well as controlling the in vivo infection progression. Interestingly, chelating agents also potentiate the activity of classical antimicrobial compounds. The differences between the microorganism and host in terms of the behavior displayed in the presence of chelating agents could provide exploitable targets for the development of an effective chemotherapy for these diseases. Consequently, metal chelators represent a novel group of antimicrobial agents with potential therapeutic applications. This review will focus on the anti-fungal and anti-protozoan action of the most common chelating agents, deciphering and discussing their mode of action.

Detection of matrix metallopeptidase-9-like proteins in Trypanosoma cruzi

In this study, the cell-associated and extracellular peptidases of Trypanosoma cruzi grown in modified Roitmans complex (MRC) medium were analyzed by measuring peptidase activity in gelatin-containing zymograms. Our results showed that the cell-associated peptidases as well as peptidases extracellularly released by T. cruzi displayed two distinct proteolytic classes: cysteine and metallopeptidase activities. The major cysteine peptidase, cruzipain, synthesized by T. cruzi cells was detected in cellular parasite content, as a 50kDa reactive polypeptide, after probing with anti-cruzipain antibody. In addition, metallo-type peptidases belonging to the matrix metallopeptidase-9 (MMP-9) family were revealed, after Western blotting, as a 97kDa protein band in cellular extract and an 85kDa polypeptide in both cellular and secreted parasite extracts. The MMP-9-like activity present in cells and spent culture medium was immunoprecipitated by an anti-MMP-9 polyclonal antibody. The surface location of MMP-9-like proteins in T. cruzi was also evidenced by means of flow cytometry analysis. Furthermore, doxycycline that has direct MMP-9 inhibiting properties in vitro, inhibited MMP-9-like activities in gel zymography, immunoprecipitation and flow cytometry analyses. This is the first report of the presence of MMP-9-like molecules in T. cruzi. The presence of a matrix extracellular-degrading enzyme may play a role in the T. cruzi-host cell interaction, making this enzyme a potential target for future drug development against this pathogenic trypanosomatid.

Candida haemulonii complex: species identification and antifungal susceptibility profiles of clinical isolates from Brazil

OBJECTIVES The emerging fungal pathogens comprising the Candida haemulonii complex (Candida haemulonii, Candida haemulonii var. vulnera and Candida duobushaemulonii) are notable for their antifungal resistance. Twelve isolates with phenotypic similarity to C. haemulonii were recovered from patients in Brazilian hospitals. Here we aimed to identify these isolates by a molecular approach, using the current classification of this fungal complex, and to evaluate their antifungal susceptibility profiles. METHODS The fungal isolates were rechecked to certify their authentication by mycology methodologies and then characterized by ITS1-5.8S-ITS2 gene sequencing. A susceptibility assay was performed using the broth microdilution method published by CLSI (M27-A3/M27-S3). RESULTS Based on biochemical tests, all Brazilian isolates were identified as C. haemulonii. After employing ITS sequencing, five isolates were identified as C. haemulonii, four as C. duobushaemulonii and three as C. haemulonii var. vulnera. All 12 clinical isolates were resistant to amphotericin B (MICs ranged from 2 to >16 mg/L) and fluconazole (MICs ≥ 64 mg/L). One isolate of C. haemulonii var. vulnera and two isolates of C. duobushaemulonii were susceptible-dose dependent to itraconazole, while the remaining isolates (75%) were resistant to this antifungal. Eight out of 12 isolates (66.7%) were resistant to voriconazole (MICs ≥ 16 mg/L), while all isolates were susceptible to caspofungin (MICs ≤ 0.5 mg/L). CONCLUSIONS Our results reinforce the importance of molecular identification in differentiating species of the C. haemulonii complex. Moreover, the antifungal multiresistant profile of clinical isolates of the C. haemulonii complex represents a challenge to the treatment of such infections.

Aspartic Peptidases of Human Pathogenic Trypanosomatids: Perspectives and Trends for Chemotherapy

Aspartic peptidases are proteolytic enzymes present in many organisms like vertebrates, plants, fungi, protozoa and in some retroviruses such as human immunodeficiency virus (HIV). These enzymes are involved in important metabolic processes in microorganisms/virus and play major roles in infectious diseases. Although few studies have been performed in order to identify and characterize aspartic peptidase in trypanosomatids, which include the etiologic agents of leishmaniasis, Chagas’ disease and sleeping sickness, some beneficial properties of aspartic peptidase inhibitors have been described on fundamental biological events of these pathogenic agents. In this context, aspartic peptidase inhibitors (PIs) used in the current chemotherapy against HIV (e.g., amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) were able to inhibit the aspartic peptidase activity produced by different species of Leishmania. Moreover, the treatment of Leishmania promastigotes with HIV PIs induced several perturbations on the parasite homeostasis, including loss of the motility and arrest of proliferation/growth. The HIV PIs also induced an increase in the level of reactive oxygen species and the appearance of irreversible morphological alterations, triggering parasite death pathways such as programed cell death (apoptosis) and uncontrolled autophagy. The blockage of physiological parasite events as well as the induction of death pathways culminated in its incapacity to adhere, survive and escape of phagocytic cells. Collectively, these results support the data showing that parasites treated with HIV PIs have a significant reduction in the ability to cause in vivo infection. Similarly, the treatment of Trypanosoma cruzi cells with pepstatin A showed a significant inhibition on both aspartic peptidase activity and growth as well as promoted several and irreversible morphological changes. These studies indicate that aspartic peptidases can be promising targets in trypanosomatid cells and aspartic proteolytic inhibitors can be benefic chemotherapeutic agents against these human pathogenic microorganisms.