Identification, Biochemical Characterization, and In Vivo Detection of a Zn-Metalloprotease with Collagenase Activity from Mannheimia haemolytica A2.

Respiratory diseases in ruminants are a main cause of economic losses to farmers worldwide. Approximately 25% of ruminants experience at least one episode of respiratory disease during the first year of life. Mannheimia haemolytica is the main etiological bacterial agent in the ruminant respiratory disease complex. M. haemolytica can secrete several virulence factors, such as leukotoxin, lipopolysaccharide, and proteases, that can be targeted to treat infections. At present, little information has been reported on the secretion of M. haemolytica A2 proteases and their host protein targets. Here, we obtained evidence that M. haemolytica A2 proteases promote the degradation of hemoglobin, holo-lactoferrin, albumin, and fibrinogen. Additionally, we performed biochemical characterization for a specific 110 kDa Zn-dependent metalloprotease (110-Mh metalloprotease). This metalloprotease was purified through ion exchange chromatography and characterized using denaturing and chaotropic agents and through zymography assays. Furthermore, mass spectrometry identification and 3D modeling were performed. Then, antibodies against the 110 kDa-Mh metalloprotease were produced, which achieved great inhibition of proteolytic activity. Finally, the antibodies were used to perform immunohistochemical tests on postmortem lung samples from sheep with suggestive histology data of pneumonic mannheimiosis. Taken together, our results strongly suggest that the 110-Mh metalloprotease participates as a virulence mechanism that promotes damage to host tissues.

Bovine holo-lactoferrin inhibits migration and invasion in MDA-MB-231 breast cancer cells.

PURPOSE: Breast cancer is the most common malignancy in developed countries and the main cause of deaths in women worldwide. Lactoferrin (Lf) is an iron-binding protein constituted for a single polypeptide chain that is folded into two symmetrical lobes that bind Fe2+ or Fe3+. Lf has the ability to reversibly bind Fe3+ and is found free of Fe3+ (Apo-Lf) or associated with Fe3+ (Holo-Lf) with a different three-dimensional conformation. However, the role of bovine Apo-Lf (Apo-BLf) and bovine Holo-Lf (Holo-BLf) in the migration and invasion induced by linoleic acid (LA) and fetal bovine serum (FBS), as well as in the expression of mesenchymal and epithelial proteins in breast cancer cells has not been studied. METHODS AND RESULTS: Scratch wound assays demonstrated that Holo-BLf and Apo-BLf do not induce migration, however they differentially inhibit the migration induced by FBS and LA in breast cancer cells MDA-MB-231. Western blot, invasion, zymography and immunofluorescence confocal microscopy assays demonstrated that Holo-BLf partly inhibit the invasion, FAK phosphorylation at tyrosine (Tyr)-397 and MMP-9 secretion, whereas it increased the number and size of focal adhesions induced by FBS in MDA-MB-231 cells. Moreover, Holo-BLf induced a slight increase of E-cadherin expression in MCF-7 cells, and inhibited vimentin expression in MCF-7 and MDA-MB-231 breast cancer cells. CONCLUSION: Holo-BLf inhibits cellular processes that mediate the invasion process in breast cancer cells.

Intestinal amoebiasis: 160 years of its first detection and still remains as a health problem in developing countries.

Amoebiasis is a parasitic disease caused by Entamoeba histolytica (E. histolytica), an extracellular enteric protozoan. This infection mainly affects people from developing countries with limited hygiene conditions, where it is endemic. Infective cysts are transmitted by the fecal-oral route, excysting in the terminal ileum and producing invasive trophozoites (amoebae). E. histolytica mainly lives in the large intestine without causing symptoms; however, possibly as a result of so far unknown signals, the amoebae invade the mucosa and epithelium causing intestinal amoebiasis. E. histolytica possesses different mechanisms of pathogenicity for the adherence to the intestinal epithelium and for degrading extracellular matrix proteins, producing tissue lesions that progress to abscesses and a host acute inflammatory response. Much information has been obtained regarding the virulence factors, metabolism, mechanisms of pathogenicity, and the host immune response against this parasite; in addition, alternative treatments to metronidazole are continually emerging. An accesible and low-cost diagnostic method that can distinguish E. histolytica from the most nonpathogenic amoebae and an effective vaccine are necessary for protecting against amoebiasis. However, research about the disease and its prevention has been a challenge due to the relationship between E. histolytica and the host during the distinct stages of the disease is multifaceted. In this review, we analyze the interaction between the parasite, the human host, and the colon microbiota or pathogenic microorganisms, which together give rise to intestinal amoebiasis.

Effect of apo-lactoferrin on leukotoxin and outer membrane vesicles of Mannheimia haemolytica A2.

Mannheimia haemolytica serotype A2 is the principal cause of pneumonic mannheimiosis in ovine and caprine livestock; this disease is a consequence of immune suppression caused by stress and associated viruses and is responsible for significant economic losses in farm production worldwide. Gram-negative bacteria such as M. haemolytica produce outer membrane (OM)-derived spherical structures named outer membrane vesicles (OMVs) that contain leukotoxin and other biologically active virulence factors. In the present study, the relationship between M. haemolytica A2 and bovine lactoferrin (BLf) was studied. BLf is an 80 kDa glycoprotein that possesses bacteriostatic and bactericidal properties and is part of the mammalian innate immune system. Apo-BLf (iron-free) showed a bactericidal effect against M. haemolytica A2, with an observed minimal inhibitory concentration (MIC) of 16 µM. Sublethal doses (2-8 µM) of apo-BLf increased the release of OMVs, which were quantified by flow cytometry. Apo-BLf modified the normal structure of the OM and OMVs, as observed through transmission electron microscopy. Apo-BLf also induced lipopolysaccharide (LPS) release from bacteria, disrupting OM permeability and functionality, as measured by silver staining and SDS and polymyxin B cell permeability assays. Western blot results showed that apo-BLf increased the secretion of leukotoxin in M. haemolytica A2 culture supernatants, possibly through its iron-chelating activity. In contrast, holo-BLf (with iron) did not have this effect, possibly due to differences in the tertiary structure between these proteins. In summary, apo-BLf affected the levels of several M. haemolytica virulence factors and could be evaluated for use in animals as an adjuvant in the treatment of ovine mannheimiosis.

Lactoferrin and Its Derived Peptides: An Alternative for Combating Virulence Mechanisms Developed by Pathogens.

Due to the emergence of multidrug-resistant pathogens, it is necessary to develop options to fight infections caused by these agents. Lactoferrin (Lf) is a cationic nonheme multifunctional glycoprotein of the innate immune system of mammals that provides numerous benefits. Lf is bacteriostatic and/or bactericidal, can stimulate cell proliferation and differentiation, facilitate iron absorption, improve neural development and cognition, promote bone growth, prevent cancer and exert anti-inflammatory and immunoregulatory effects. Lactoferrin is present in colostrum and milk and is also produced by the secondary granules of polymorphonuclear leukocytes, which store this glycoprotein and release it at sites of infection. Lf is also present in many fluids and exocrine secretions, on the surfaces of the digestive, respiratory and reproductive systems that are commonly exposed to pathogens. Apo-Lf (an iron-free molecule) can be microbiostatic due to its ability to capture ferric iron, blocking the availability of host iron to pathogens. However, apo-Lf is mostly microbicidal via its interaction with the microbial surface, causing membrane damage and altering its permeability function. Lf can inhibit viral entry by binding to cell receptors or viral particles. Lf is also able to counter different important mechanisms evolved by microbial pathogens to infect and invade the host, such as adherence, colonization, invasion, production of biofilms and production of virulence factors such as proteases and toxins. Lf can also cause mitochondrial and caspase-dependent regulated cell death and apoptosis-like in pathogenic yeasts. All of these mechanisms are important targets for treatment with Lf. Holo-Lf (the iron-saturated molecule) can contain up to two ferric ions and can also be microbicidal against some pathogens. On the other hand, lactoferricins (Lfcins) are peptides derived from the N-terminus of Lf that are produced by proteolysis with pepsin under acidic conditions, and they cause similar effects on pathogens to those caused by the parental Lf. Synthetic analog peptides comprising the N-terminus Lf region similarly exhibit potent antimicrobial properties. Importantly, there are no reported pathogens that are resistant to Lf and Lfcins; in addition, Lf and Lfcins have shown a synergistic effect with antimicrobial and antiviral drugs. Due to the Lf properties being microbiostatic, microbicidal, anti-inflammatory and an immune modulator, it represents an excellent natural alternative either alone or as adjuvant in the combat to antibiotic multidrug-resistant bacteria and other pathogens. This review aimed to evaluate the data that appeared in the literature about the effects of Lf and its derived peptides on pathogenic bacteria, protozoa, fungi and viruses and how Lf and Lfcins inhibit the mechanisms developed by these pathogens to cause disease.

The Impact of Lactoferrin on the Growth of Intestinal Inhabitant Bacteria.

Lactoferrin (Lf) is an iron-binding milk glycoprotein that promotes the growth of selected probiotic strains. The effect of Lf on the growth and diversification of intestinal microbiota may have an impact on several issues, including (i) strengthening the permeability of the epithelial cell monolayer, (ii) favoring the microbial antagonism that discourages the colonization and proliferation of enteric pathogens, (iii) enhancing the growth and maturation of cell-monolayer components and gut nerve fibers, and (iv) providing signals to balance the anti- and pro-inflammatory responses resulting in gut homeostasis. Given the beneficial role of probiotics, this contribution aims to review the current properties of bovine and human Lf and their derivatives in in vitro probiotic growth and Lf interplay with microbiota described in the piglet model. By using Lf as a component in pharmacological products, we may enable novel strategies that promote probiotic growth while conferring antimicrobial activity against multidrug-resistant microorganisms that cause life-threatening diseases, especially in neonates.

Iron responsive-like elements in the parasite Entamoeba histolytica.

In Entamoeba histolytica, iron modulates virulence and gene expression via unknown regulatory mechanisms. The existence of a posttranscriptional iron regulatory system parallel with the iron-responsive element (IRE)/iron regulatory protein (IRP) system in the protozoan Trichomonas vaginalis has recently been reported. Due to their evolutionary closeness and the importance of iron for growth and virulence in these protozoa, we hypothesized the existence of an IRE/IRP-like mechanism in E. histolytica. To determine the presence of IRE-like elements in some mRNAs from this parasite, we performed in silico analyses of the 5'- and 3'-UTRs of mRNAs encoding virulence factors and cytoskeleton, ribosomal and metabolism proteins. The Zuker mfold software predicted IRE-like secondary structures in 52 of the 135 mRNAs analysed. However, only nine structures shared sequence similarity with the apical loop sequence (CAGUGN) of the previously reported human IRE-ferritin, whereas the GUU/UUG protozoan-specific motif was detected in 23 stem-loop structures. A new motif, AUU/AUUU, was also observed in 23 structures, suggesting the possible existence of an amoeba-specific motif. Additionally, cross-linking and RNA electrophoretic mobility shift assays showed specific RNA-protein interactions, using as a model two amoebic IRE-like elements from iron-regulated mRNAs and HeLa, T. vaginalis and E. histolytica cytoplasmic proteins. Our data suggest the presence of a posttranscriptional iron regulatory IRE/IRP-like mechanism in E. histolytica.

Mannheimia haemolytica A2 secretes different proteases into the culture medium and in outer membrane vesicles.

Respiratory diseases in ruminants have a significantly negative impact on the worldwide economy. The bacterium Mannheimia haemolytica is involved in pneumonic infections in bovine and ovine. In gram-negative bacteria, six secretion systems related to the colonization process and host tissue damage have been reported. In addition, in the last two decades, the production of outer membrane vesicles has been studied as a different bacterial strategy to release virulence factors, such as exotoxins, lipopolysaccharides, and proteases. However, in M. haemolytica serotype A2, protease secretion and release in vesicles have not been reported as virulence mechanisms. The aim of this work was to identify proteases released into the culture supernatant and in vesicles of M. haemolytica A2. Our results showed evident differences in the molecular mass and activity of proteases present in culture supernatants and outer membrane vesicles based on zymography assays. The biochemical characterization of M. haemolytica proteases revealed that the main types were cysteine and metalloproteases. A specific metalloprotease of 100 kDa was active in the culture supernatants, but it was not active and was found in low quantities in vesicles. Proteases could be an important virulence factor during the infectious pneumonic process led by M. haemolytica.

Lactoferrin: Balancing Ups and Downs of Inflammation Due to Microbial Infections.

Lactoferrin (Lf) is a glycoprotein of the primary innate immune-defense system of mammals present in milk and other mucosal secretions. This protein of the transferrin family has broad antimicrobial properties by depriving pathogens from iron, or disrupting their plasma membranes through its highly cationic charge. Noteworthy, Lf also exhibits immunomodulatory activities performing up- and down-regulation of innate and adaptive immune cells, contributing to the homeostasis in mucosal surfaces exposed to myriad of microbial agents, such as the gastrointestinal and respiratory tracts. Although the inflammatory process is essential for the control of invasive infectious agents, the development of an exacerbated or chronic inflammation results in tissue damage with life-threatening consequences. In this review, we highlight recent findings in in vitro and in vivo models of the gut, lung, oral cavity, mammary gland, and liver infections that provide experimental evidence supporting the therapeutic role of human and bovine Lf in promoting some parameters of inflammation and protecting against the deleterious effects of bacterial, viral, fungal and protozoan-associated inflammation. Thus, this new knowledge of Lf immunomodulation paves the way to more effective design of treatments that include native or synthetic Lf derivatives, which may be useful to reduce immune-mediated tissue damage in infectious diseases.

Two outer membrane proteins are bovine lactoferrin-binding proteins in Mannheimia haemolytica A1.

Mannheimia haemolytica is a Gram negative bacterium that is part of the bovine respiratory disease, which causes important economic losses in the livestock industry. In the present work, the interaction between M. haemolytica A1 and bovine lactoferrin (BLf) was studied. This iron-chelating glycoprotein is part of the mammalian innate-immune system and is present in milk and mucosal secretions; Lf is also contained in neutrophils secondary granules, which release this glycoprotein at infection sites. It was evidenced that M. haemolytica was not able to use iron-charged BLf (BholoLf) as a sole iron source; nevertheless, iron-lacked BLf (BapoLf) showed a bactericidal effect against M. haemolytica with MIC of 4.88 ± 1.88 and 7.31 ± 1.62 µM for M. haemolytica strain F (field isolate) and M. haemolytica strain R (reference strain), respectively. Through overlay assays and 2-D electrophoresis, two OMP of 32.9 and 34.2 kDa with estimated IP of 8.18 and 9.35, respectively, were observed to bind both BapoLf and BholoLf; these OMP were identified by Maldi-Tof as OmpA (heat-modifiable OMP) and a membrane protein (porin). These M. haemolytica BLf binding proteins could be interacting in vivo with both forms of BLf depending on the iron state of the bovine.

Acanthamoeba castellanii Proteases are Capable of Degrading Iron-Binding Proteins as a Possible Mechanism of Pathogenicity.

Acanthamoeba castellanii, a free-living amoeba, is an amphizoic organism that can behave as an opportunistic pathogen, causing granulomatous amoebic encephalitis in immunocompromised patients or infecting immunocompetent individuals via cutaneous lesions, sinusoidal infections, or amoebic keratitis. Therefore, this amoeba could be in contact with different iron-binding proteins, such as lactoferrin in tears and mucosa and transferrin and hemoglobin in blood. Iron is a vital and necessary element for host metabolism but also for parasite survival. Accordingly, parasites have developed iron uptake mechanisms, one of which is the utilization of proteases to degrade host iron-binding proteins. In this work, we performed a partial biochemical characterization of A. castellanii proteases at different pHs and utilizing protease inhibitors with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and copolymerized with different iron-binding proteins. We describe for the first time the presence of several cysteine proteases in a total A. castellanii crude extract and in conditioned culture medium precipitated with ethanol. These amoebic peptidases degraded human holo-lactoferrin, holo-transferrin, hemoglobin, and horse spleen ferritin; some of these proteases were substrate specific, and others degraded multiple substrates. These proteases could be considered virulence factors that promote iron acquisition from the host.

Effect of bovine apo-lactoferrin on the growth and virulence of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae (App) is a Gram-negative bacterium that causes porcine pleuropneumonia, leading to economic losses in the swine industry. Due to bacterial resistance to antibiotics, new treatments for this disease are currently being sought. Lactoferrin (Lf) is an innate immune system glycoprotein of mammals that is microbiostatic and microbicidal and affects several bacterial virulence factors. The aim of this study was to investigate whether bovine iron-free Lf (BapoLf) has an effect on the growth and virulence of App. Two serotype 1 strains (reference strain S4074 and the isolate BC52) and a serotype 7 reference strain (WF83) were analyzed. First, the ability of App to grow in iron-charged BLf was discarded because in vivo, BapoLf sequesters iron and could be a potential source of this element favoring the infection. The minimum inhibitory concentration of BapoLf was 14.62, 11.78 and 10.56 µM for the strain BC52, S4074 and WF83, respectively. A subinhibitory concentration (0.8 µM) was tested by assessing App adhesion to porcine buccal epithelial cells, biofilm production, and the secretion and function of toxins and proteases. Decrease in adhesion (24-42 %) was found in the serotype 1 strains. Biofilm production decreased (27 %) for only the strain 4074 of serotype 1. Interestingly, biofilm was decreased (60-70 %) in the three strains by BholoLf. Hemolysis of erythrocytes and toxicity towards HeLa cells were not affected by BapoLf. In contrast, proteolytic activity in all strains was suppressed in the presence of BapoLf. Finally, oxytetracycline produced synergistic effect with BapoLf against App. Our results suggest that BapoLf affects the growth and several of the virulence factors in App.

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.

Lactoferrin and Lysozyme Inhibit the Proteolytic Activity and Cytopathic Effect of Naegleria fowleri Enzymes.

Naegleria fowleri is a ubiquitous free-living amoeba that causes primary amoebic meningoencephalitis. As a part of the innate immune response at the mucosal level, the proteins lactoferrin (Lf) and lysozyme (Lz) are secreted and eliminate various microorganisms. We demonstrate that N. fowleri survives the individual and combined effects of bovine milk Lf (bLf) and chicken egg Lz (cLz). Moreover, amoebic proliferation was not altered, even at 24 h of co-incubation with each protein. Trophozoites' ultrastructure was evaluated using transmission electron microscopy, and these proteins did not significantly alter their organelles and cytoplasmic membranes. Protease analysis using gelatin-zymograms showed that secreted proteases of N. fowleri were differentially modulated by bLf and cLz at 3, 6, 12, and 24 h. The bLf and cLz combination resulted in the inhibition of N. fowleri-secreted proteases. Additionally, the use of protease inhibitors on bLf-zymograms demonstrated that secreted cysteine proteases participate in the degradation of bLf. Nevertheless, the co-incubation of trophozoites with bLf and/or cLz reduced the cytopathic effect on the MDCK cell line. Our study suggests that bLf and cLz, alone or together, inhibited secreted proteases and reduced the cytopathic effect produced by N. fowleri; however, they do not affect the viability and proliferation of the trophozoites.

Iron-saturated bovine lactoferrin: a promising chemopreventive agent for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a tumor with minimal chance of cure due to underlying liver diseases, late diagnosis, and inefficient treatments. Thus, HCC treatment warrants the development of additional strategies. Lactoferrin (Lf) is a mammalian multifunctional iron-binding glycoprotein of the innate immune response and can be found as either a native low iron form (native-Lf) or a high iron form (holo-Lf). Bovine Lf (bLf), which shares many functions with human Lf (hLf), is safe for humans and has several anticancer activities, including chemotherapy boost in cancer. We found endogenous hLf is downregulated in HCC tumors compared with normal liver, and decreased hLf levels in HCC tumors are associated with shorter survival of HCC patients. However, the chemoprotective effect of 100% iron saturated holo-bLf on experimental hepatocarcinogenesis has not yet been determined. We aimed to evaluate the chemopreventive effects of holo-bLf in different HCC models. Remarkably, a single dose (200 mg kg-1) of holo-bLf was effective in preventing early carcinogenic events in a diethylnitrosamine induced HCC in vivo model, such as necrosis, ROS production, and the surge of facultative liver stem cells, and eventually, holo-bLf reduced the number of preneoplastic lesions. For an established HCC model, holo-bLf treatment significantly reduced HepG2 tumor burden in xenotransplanted mice. Finally, holo-bLf in combination with sorafenib, the advanced HCC first-line treatment, synergistically decreased HepG2 viability by arresting cells in the G0/G1 phase of the cell cycle. Our findings provide the first evidence suggesting that holo-bLf has the potential to prevent HCC or to be used in combination with treatments for established HCC.

A model of alcoholic liver disease based on different hepatotoxics leading to liver cancer.

The worst-case scenario related to alcoholic liver disease (ALD) arises after a long period of exposure to the harmful effect of alcohol consumption along with other hepatotoxics. ALD encompasses a broad spectrum of liver-associated disorders, such as steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Based on the chronic administration of different hepatotoxics, including ethanol, sucrose, lipopolysaccharide, and low doses of diethylnitrosamine over a short period, here we aimed to develop a multiple hepatotoxic (MHT)-ALD model in the mouse that recapitulates the human ALD-associated disorders. We demonstrated that the MHT-ALD model induces ADH1A and NXN, an ethanol metabolizer and a redox-sensor enzyme, respectively; promotes steatosis associated with the induction of the lipid droplet forming FSP27, inflammation identified by the infiltration of hepatic neutrophils-positive to LY-6G marker, and the increase of MYD88 level, a protein involved in inflammatory response; and stimulates the early appearance of cellular senescence identified by the senescence markers SA-ß-gal activity and p-H2A.XSer139. It also induces fibrosis associated with increased desmin, a marker of hepatic stellate cells whose activation leads to the deposition of collagen fibers, accompanied by cell death and compensatory proliferation revealed by increased CASP3-mediated apoptosis, and KI67- and PCNA-proliferation markers, respectively. It also induces histopathological traits of malignancy and the level of the HCC marker, GSTP1. In conclusion, we provide a useful model for exploring the chronological ALD-associated alterations and stages, and addressing therapeutic approaches.

Hemoglobin uptake and utilization by human protozoan parasites: a review.

The protozoan disease is a major global health concern. Amoebiasis, leishmaniasis, Chagas disease, and African sleeping sickness affect several million people worldwide, leading to millions of deaths annually and immense social and economic problems. Iron is an essential nutrient for nearly all microbes, including invading pathogens. The majority of iron in mammalian hosts is stored intracellularly in proteins, such as ferritin and hemoglobin (Hb). Hb, present in blood erythrocytes, is a very important source of iron and amino acids for pathogenic microorganisms ranging from bacteria to eukaryotic pathogens, such as worms, protozoa, yeast, and fungi. These organisms have developed adequate mechanisms to obtain Hb or its byproducts (heme and globin) from the host. One of the major virulence factors identified in parasites is parasite-derived proteases, essential for host tissue degradation, immune evasion, and nutrient acquisition. The production of Hb-degrading proteases is a Hb uptake mechanism that degrades globin in amino acids and facilitates heme release. This review aims to provide an overview of the Hb and heme-uptake mechanisms utilized by human pathogenic protozoa to survive inside the host.

Acanthamoeba castellanii Genotype T4: Inhibition of Proteases Activity and Cytopathic Effect by Bovine Apo-Lactoferrin.

Acanthamoeba castellanii genotype T4 is a clinically significant free-living amoeba that causes granulomatous amoebic encephalitis and amoebic keratitis in human beings. During the initial stages of infection, trophozoites interact with various host immune responses, such as lactoferrin (Lf), in the corneal epithelium, nasal mucosa, and blood. Lf plays an important role in the elimination of pathogenic microorganisms, and evasion of the innate immune response is crucial in the colonization process. In this study, we describe the resistance of A. castellanii to the microbicidal effect of bovine apo-lactoferrin (apo-bLf) at different concentrations (25, 50, 100, and 500 µM). Acanthamoeba castellanii trophozoites incubated with apo-bLf at 500 µM for 12 h maintained 98% viability. Interestingly, despite this lack of effect on viability, our results showed that the apo-bLf inhibited the cytopathic effect of A. castellanii in MDCK cells culture, and analysis of amoebic proteases by zymography showed significant inhibition of cysteine and serine proteases by interaction with the apo-bLf. From these results, we conclude that bovine apo-Lf influences the activity of A. castellanii secretion proteases, which in turn decreases amoebic cytopathic activity.

Oral lactoferrin treatment resolves amoebic intracecal infection in C3H/HeJ mice.

Entamoeba histolytica is a protozoan parasite that causes amoebiasis, an illness that affects many people around the world. We have previously reported that lactoferrin is able to kill E. histolytica in in vitro cultures. The aim of the present study was to evaluate the therapeutic effect of orally administered bovine lactoferrin in the control of intestinal amoebiasis of susceptible C3H/HeJ mice. The results showed that 20 mg lactoferrin/kg orally administered each day for 1 week was able to eliminate the infection in 63% of the mice, since neither trophozoites nor evidence of epithelial damage and (or) swelling were found in tissue sections of the cecum. The rest of the treated animals (37%) showed a decrease in trophozoite numbers and mucus secreted to the lumen, as compared with untreated and infected mice (p < 0.05). By immunohistochemistry, the profile of secreted cytokines in the cecum revealed that infected but untreated animals showed a mixed Th1/regulatory cytokines profile, whereas the cecum of mice treated (cured) showed a Th2 cytokine profile (IL-4) and expression of the multifunctional IL-6. In addition, cytokines and increasing cecal production of total IgA antibodies were found associated with little inflammation and disease control observed in the cecum of lactoferrin-treated animals. These results suggest that oral administration of lactoferrin can control intestinal amoebic infection probably by killing amoebas or favoring their removal and reestablish the antiinflammatory intestinal environment.