Catalases in the pathogenesis of Sporothrix schenckii research.

Pathogenic fungal infection success depends on the ability to escape the immune response. Most strategies for fungal infection control are focused on the inhibition of virulence factors and increasing the effectiveness of antifungal drugs. Nevertheless, little attention has been focused on their physiological resistance to the host immune system. Hints may be found in pathogenic fungi that also inhabit the soil. In nature, the saprophyte lifestyle of fungi is also associated with predators that can induce oxidative stress upon cell damage. The natural sources of nutrients for fungi are linked to cellulose degradation, which in turn generates reactive oxygen species (ROS). Overall, the antioxidant arsenal needed to thrive both in free-living and pathogenic lifestyles in fungi is fundamental for success. In this review, we present recent findings regarding catalases and oxidative stress in fungi and how these can be in close relationship with pathogenesis. Additionally, special focus is placed on catalases of Sporothrix schenckii as a pathogenic model with a dual lifestyle. It is assumed that catalase expression is activated upon exposure to H2O2, but there are reports where this is not always the case. Additionally, it may be relevant to consider the role of catalases in S. schenckii survival in the saprophytic lifestyle and why their study can assess their involvement in the survival and therefore, in the virulence phenotype of different species of Sporothrix and when each of the three catalases are required. Also, studying antioxidant mechanisms in other isolates of pathogenic and free-living fungi may be linked to the virulence phenotype and be potential therapeutic and diagnostic targets. Thus, the rationale for this review to place focus on fungal catalases and their role in pathogenesis in addition to counteracting the effect of immune system reactive oxygen species. Fungi that thrive in soil and have mammal hosts could shed light on the importance of these enzymes in the two types of lifestyles. We look forward to encouraging more research in a myriad of areas on catalase biology with a focus on basic and applied objectives and placing these enzymes as virulence determinants.

Trichomonas vaginalis triggers neutrophil extracellular traps reducing parasite integrity and growth.

Trichomoniasis-caused by the parasite Trichomonas vaginalis-is associated with a high inflammatory process that may contribute to the risk of suffering from other medical complications. Our study focused on the in vitro interaction of T. vaginalis with human neutrophils because these are the most abundant cells implicated in the characteristic inflammatory process of trichomoniasis. This study showed that T. vaginalis and its surface glycoconjugates (lipophosphoglycan and/or lipoglycan) induced the formation of human neutrophil extracellular traps (NETs). After the trichomonad-neutrophil interaction, parasite integrity was at 32.9%, and the subsequent parasite growth was at 35.2% compared to those of control trophozoites (100%) incubated under the same conditions without neutrophils. In the presence of an antibody against the TLR-4 receptor, DNase I or micrococcal nuclease (MNase), neutrophils reduced the DNA fibres of the NETs and the amount of extracellular DNA, allowing a higher subsequent growth of T. vaginalis, at 52% with the anti-TLR-4 antibody and 62.6% with the enzymes. These results indicated that T. vaginalis induced the formation of extracellular traps by human neutrophils and, because of the interaction with neutrophils and NETs, parasite integrity and growth decreased.

Contribution of neutral sphingomyelinases to in vitro virulence of Entamoeba histolytica.

Amoebiasis is a worldwide health problem caused by the pathogen Entamoeba histolytica. Several virulence factors have been implicated in host invasion, immune evasion, and tissue damage. There are still new factors that remain to be elucidated and characterized. In this work, we obtained amoebic transfectants overexpressing three of the neutral sphingomyelinase enzymes encoded in the E. histolytica genome. The EhnSM3 overexpression induced an increase in hemolytic and cytotoxic activities, besides an increase in gene expression of amoebapore A, B, and C. Meanwhile the EhnSM1 and EhnSM2 overexpression caused an increase in cytopathic activity. In all the neutral sphingomyelinases overexpressing strains, the gene expression levels for cysteine proteinase 5, adhesin 112 and, heavy and light Gal/GalNAc lectin subunits were not affected. We propose that the increase of cytotoxic and lytic effect of EhnSM3 overexpressed strain can be related to the sum of the effect of EhnSM3 plus amoebapores, in a process cell contact-dependent or as mediator by inducing the gene expression of amoebapores enabling a link between EhnSM3 with the virulence phenotype in E. histolytica. Our results suggest a differential role for neutral sphingomyelinases in E. histolytica virulence.

Antibacterial and cell penetrating effects of LFcin17-30, LFampin265-284, and LF chimera on enteroaggregative Escherichia coli.

Lactoferrin (LF) is a protein with antimicrobial activity, which is conferred in part by 2 regions contained in its N-terminal lobe. These regions have been used to develop the following synthetic peptides: lactoferricin17-30, lactoferrampin265-284, and LF chimera (a fusion of lactoferricin17-30 and lactoferrampin265-284). We have reported that these LF peptides have antibacterial activity against several pathogenic bacteria; however, the exact mechanism of action has not been established. Here, we report the effects of LF peptides on the viability of enteroaggregative Escherichia coli (EAEC) and the ability of these peptides to penetrate into the bacteria cytoplasm. The viability of EAEC treated with LF peptides was determined via enumeration of colony-forming units, and the binding and internalization of the LF peptides was followed via immunogold labeling and electron microscopy. Treatment of EAEC with 20 and 40 µmol/L LF peptides reduced bacterial growth compared with untreated bacteria. Initially the peptides associated with the plasma membrane, but after 5 to 30 min of incubation, the peptides were found in the cytoplasm. Remarkably, bacteria treated with LF chimera developed cytosolic electron-dense structures that contained the antimicrobial peptide. Our results suggest that the antibacterial mechanism of LF peptides on EAEC involves their interaction with and penetration into the bacteria.

Random Transposon Mutagenesis for Cell-Envelope Resistant to Phage Infection.

In order to identify host components involved in the infective process of bacteriophages, we developed a wide-range strategy to obtain cell envelope mutants, using Escherichia coli W3110 and its specific phage mEp213. The strategy consisted in four steps: (1) random mutagenesis using transposon miniTn10Km(r); (2) selection of phage-resistant mutants by replica-plating; (3) electroporation of the phage-resistant mutants with mEp213 genome, followed by selection of those allowing phage development; and (4) sequencing of the transposon-disrupted genes. This strategy allowed us to distinguish the host factors related to phage development or multiplication within the cell, from those involved in phage infection at the level of the cell envelope.

Strategies of Intracellular Pathogens for Obtaining Iron from the Environment.

Most microorganisms are destroyed by the host tissues through processes that usually involve phagocytosis and lysosomal disruption. However, some organisms, called intracellular pathogens, are capable of avoiding destruction by growing inside macrophages or other cells. During infection with intracellular pathogenic microorganisms, the element iron is required by both the host cell and the pathogen that inhabits the host cell. This minireview focuses on how intracellular pathogens use multiple strategies to obtain nutritional iron from the intracellular environment in order to use this element for replication. Additionally, the implications of these mechanisms for iron acquisition in the pathogen-host relationship are discussed.

Bactericidal effect of bovine lactoferrin and synthetic peptide lactoferrin chimera in Streptococcus pneumoniae and the decrease in luxS gene expression by lactoferrin.

Streptococcus pneumoniae (pneumococcus) is responsible for nearly one million child deaths annually. Pneumococcus causes infections such as pneumonia, otitis media, meningitis, and sepsis. The human immune system includes antibacterial peptides and proteins such as lactoferrin (LF), but its activity against pneumococcus is not fully understood. The aim of this work was to evaluate the bactericidal effect of bovine lactoferrin (bLF) and the synthetic LF-peptides lactoferricin (LFcin17-30), lactoferrampin (LFampin265-284), and LFchimera against S. pneumoniae planktonic cells. The mechanism of damage was also investigated, as well as the impact of these peptides on the transcription levels of genes known to encode important virulence factors. S. pneumoniae planktonic cells were treated with bLF, LFcin17-30, LFampin265-284 and LFchimera at different time points. The viability of treated planktonic cells was assessed by dilution and plating (in CFU/ml). The interaction between LF and LF-peptides coupled to fluorescein was visualized using a confocal microscope and flow cytometry, whereas the damage at structural levels was observed by electron microscopy. Damage to bacterial membranes was further evaluated by membrane permeabilization by use of propidium iodide and flow cytometry, and finally, the expression of pneumococcal genes was evaluated by qRT-PCR. bLF and LFchimera were the best bactericidal agents. bLF and peptides interacted with bacteria causing changes in the shape and size of the cell and membrane permeabilization. Moreover, the luxS gene was down-regulated in bacteria treated with LF. In conclusion, LF and LFchimera have a bactericidal effect, and LF down-regulates genes involved in the pathogenicity of pneumococcus, thus demonstrating potential as new agents for the treatment of pneumococcal infections.

A novel strategy to isolate cell-envelope mutants resistant to phage infection: bacteriophage mEp213 requires lipopolysaccharides in addition to FhuA to enter Escherichia coli K-12.

We have developed a direct and efficient strategy, based on a three-step method, to select bacterial cell-envelope mutants resistant to bacteriophage infection. Escherichia coli K-12 strain W3110 underwent classical transposon mutagenesis followed by replica plating and selection for mutants resistant to infection by coliphage mEp213. To verify that phage resistance was due to mutations in the cell envelope, we transformed host cells with the viral genome using electroporation and selected those in which virions were subsequently detected in the supernatant. Among the nine mutants resistant to coliphage infection that we selected, six were in the fhuA gene, two were mutated in the waaC gene, and one was mutated in the gmhD gene. The latter two gene products are involved in the synthesis of lipopolysaccharide (LPS). The efficiency of plating and adsorption of phage mEp213 was affected in these mutants. We verified that LPS is required for the efficient infection of phage λ as well. We propose that this mutation-and-selection strategy can be used to find host factors involved in the initial steps of phage infection for any cognate pair of phage and bacteria.

Overexpression of Ipe protein from the coliphage mEp021 induces pleiotropic effects involving haemolysis by HlyE-containing vesicles and cell death.

Lysogenic Escherichia coli K-12 harbouring the prophage mEp021 displays haemolytic activity. From a genomic library of mEp021, we identified an open reading frame (ORF 4) that was responsible for the haemolytic activity. However, the ORF 4 sequence contains four initiation codons in the same frame: ORF 4.1-ORF 4.4, coding for 83-a.a., 82-a.a., 77-a.a. and 72-a.a. products, respectively. The expression of the cloned ORF 4.3, or inducer of pleiotropic effects (ipe), reproduced the haemolytic phenotype in a native strain carrying the gene hlyE(+), but not in the mutant hlyE(-) strain. The overexpression of Ipe induced several pleiotropic effects, such as the inhibition of cell growth and the deregulation of cell division, which resulted in a mixture of normal and desiccated-like cells: normal-filamentous, desiccated-like-filamentous bacilli, minicells etc. Other effects included abnormalities of the cell membrane, the production of vesicles containing HlyE, and finally, cell death. These events were analysed at the molecular level by microarray assays. The global transcription profile of E. coli K-12 strain MC4100, which expressed Ipe after 4 h, revealed differential expression of various genes, most of which were related either to cell membrane and murein biosynthesis or to cell division. The up-regulation of some of these transcripts was confirmed by qRT-PCR. Additional research is needed to determine whether these effects are directly related to Ipe activity or are consequences of the cellular responses to putative structural damage induced by Ipe.

Identification of Actinobacillus pleuropneumoniae biovars 1 and 2 in pigs using a PCR assay.

Actinobacillus pleuropneumoniae causes swine pleuropneumonia worldwide. Previously, we described a gene sequence of approximately 800bp in A. pleuropneumoniae serotype 1 that encodes a metalloprotease of 24kDa, (Genbank accession no. AY217757). We selected primers carrying the forward and reverse 5'-terminal sequences of this region of the gene for the development of a species-specific PCR assay. The primers amplified an 800bp sequence from isolated DNA and lysed bacteria of the 13 A. pleuropneumoniae biovar 1 serotypes, with the exception of subtype 1b. The primers also amplified the sequence in nasal secretion cultures from pigs with chronic and acute experimental pleuropneumonia. No PCR products were detected when A. pleuropneumoniae serotypes of biovar 2 were used. Internal primers from this gene sequence detected biovar 2 and subtype 1b, leading to the production of a 350bp PCR product. The primers did not amplify DNA from other related species from the Pasteurellaceae family. The 800bp PCR assay was sensitive in vitro, with a detection limit of 5.5pg of extracted DNA, and an average of 120CFU. The specificity and sensitivity of this PCR assay make it a useful method for the rapid identification and diagnosis of A. pleuropneumoniae.

Prefrontal and tegmental electrical activity during olfactory stimulation in virgin and lactating rats.

This study was designed to analyze if the electroencephalographic (EEG) activity of the medial prefrontal cortex (mPFC) and the ventral tegmental area (VTA) was modified throughout various reproductive stages of female rats exposed to pup-associated or non-associated odors. Simultaneous EEG recordings were obtained from right and left mPFC and VTA in adult female rats during the smelling of nest bedding and female bedding under three reproductive states: proestrus-estrus (P-E), diestrus (D) and lactation (L). Absolute (AP) and relative (RP) powers of three EEG band frequencies, and interhemispheric correlation (r) of EEG activity were calculated and compared among conditions. During the awake-quiet condition, RP of the 12-21 Hz band was significantly higher in the right and left mPFC as well as in the right VTA of lactating rats as compared to P-E rats. During the smelling of nest bedding, the RP of the 8-11 Hz in the mPFC became increased while that of the 6-7 and 12-21 Hz decreased in the three reproductive stages. In the VTA, this phenomenon was mainly observed in lactating rats. Only the RP of the lower frequencies (6-7 and 8-11 Hz bands) was higher in the right mPFC and in the left and right VTA, respectively, of the lactating rats with respect to P-E rats, while that of the 12-21 Hz band was lower in lactating as compared to P-E rats. Moreover, the interprefrontal correlation of the lower-frequency bands was higher in relation to smelling of nest bedding in diestrus and lactating rats, whereas during the smelling of female bedding the correlation of the 6-7 Hz band was increased only in the diestrus rats as compared to P-E rats. These data indicate that EEG activity recording is a sensitive tool to study the functionality of the mPFC and VTA during different reproductive states and suggest the possible participation of these structures in the processing of olfactory stimuli associated to pups to modulate the motivational and performance processes, crucial for the expression of maternal behavior.