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 emergence of multidrug-resistant bacterial strains with respect to commercially available antimicrobial drugs has marked a watershed in treatment therapies to fight pathogens and has stimulated research on alternative remedies. Proteins of the innate immune system of mammals have been highlighted as potentially yielding possible treatment options for infections. Lactoferrin (Lf) is one of these proteins; interestingly, no resistance to it has been found. Lf is a conserved cationic nonheme glycoprotein that is abundant in milk and is also present in low quantities in mucosal secretions. Moreover, Lf is produced and secreted by the secondary granules of neutrophils at infection sites. Lf is a molecule of approximately 80 kDa that displays multiple functions, such as antimicrobial, anti-viral, anti-inflammatory, and anticancer actions. Lf can synergize with antibiotics, increasing its potency against bacteria. Lactoferricins (Lfcins) are peptides resulting from the N-terminal end of Lf by proteolytic cleavage with pepsin. They exhibit several anti-bacterial effects similar to those of the parental glycoprotein. Synthetic analog peptides exhibiting potent antimicrobial properties have been designed. The aim of this review is to update understanding of the structure and effects of Lf and Lfcins as anti-bacterial compounds, focusing on the mechanisms of action in bacteria and the use of Lf in treatment of infections in patients, including those studies where no significant differences were found. Lf could be an excellent option for prevention and treatment of bacterial diseases, mainly in combined therapies with antibiotics or other antimicrobials.

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.

Mannheimia haemolytica serotype A2 is the main bacterial causative agent of ovine mannheimiosis, a disease that leads to substantial economic losses for livestock farmers. Several virulence factors allow M. haemolytica to colonize the lungs and establish infection. Virulence factors can be directly secreted into the environment by bacteria but are also released through outer membrane vesicles (OMVs). In addition, due to the abuse of antibiotics in the treatment of this disease, multidrug-resistant bacterial strains of M. haemolytica have emerged. One therapeutic alternative to antibiotics or an adjuvant to be used in combination with antibiotics could be lactoferrin (Lf), a multifunctional cationic glycoprotein of the mammalian innate immune system to which no bacterial resistance has been reported. The aim of this work was to determine the effect of bovine iron-free Lf (apo-BLf) on the production and secretion of proteases into culture supernatant (CS) and on their release in OMVs. Zymography assays showed that addition of sub-MIC concentrations of apo-BLf to M. haemolytica cultures inhibited protease secretion without affecting culture growth. Biochemical characterization revealed that these proteases were mainly cysteine- and metalloproteases. The secretion of a 100 kDa metalloprotease was inhibited by sub-MIC concentrations of apo-BLf since this protease was present in the cytoplasm and OMVs but not in CS proteins, as corroborated by Western blotting. On the other hand, proteases produced by M. haemolytica caused cleavage of apo-BLf. However, when Lf is cleaved, peptides known as lactoferricins, which are more bactericidal than natural Lf, can be produced. M. haemolytica A2 protease-mediated degradation of host tissue proteins could be an important virulence factor during the infectious process of pneumonia in ovines. The mechanism of M. haemolytica protease secretion could be inhibited by treatment with apo-BLf in animals.

Lactic acid bacteria are components of the gastrointestinal tract microbiota in both humans and animals and are widely used as probiotics. Lactobacillus is the most closely related genus to probiotic activity. It is capable of releasing membrane microvesicles (MVs), whose primary functions include carrying and transmitting antigens to host tissues and modulating host defense responses. In the present study, MVs were isolated from Lactobacillus acidophilus resident in the ileum of free-living rats, and their immunostimulant effect was evaluated in two biological models. MVs were characterized using SDS-PAGE electrophoresis, electron microscopy, and nanoparticle tracking analysis. In the first model, the immunostimulatory effect of MVs was evaluated on ovine abomasal explants, which had been previously stimulated with MVs and then challenged with third-stage larvae of Haemonchus contortus. This resulted in a decrease in the percentage of larval association and favored the migration of inflammatory cells to the infection site. In the second model, the macrophage cell line RAW 264.7 was stimulated with MVs to evaluate the expression of transcripts encoding IL-1β and TNF-α. MVs isolated from L. acidophilus demonstrate immunostimulatory and probiotic effects in the two biological models assessed. This suggested that the MVs possess similar immunostimulatory effects as those reported for the parent bacteria.

For Entamoeba histolytica to establish an infection, it must employ several mechanisms of pathogenicity to produce and secrete virulence factors that allow the parasite to adhere to and finally colonize and invade the host. However, in the intestinal epithelium, trophozoites (amoebae) encounter lactoferrin (Lf), a glycoprotein of the first line of defense, together with immunoglobulins and other molecules. We previously reported that iron-free bovine Lf (bLf) could kill amoebae both in vitro and in animal models of intestinal and hepatic amoebiasis. In this work, selected pathogenic mechanisms were evaluated in trophozoites by exposing cultures to sublethal concentrations of bLf to determine which amoebic functions could be altered. At a sublethal bLf concentration, this glycoprotein was removed from the trophozoite. In the presence of erythrocytes, bLf colocalized with erythrocytes at the capping site; this was observed by confocal microscopy of living cells. In addition, the erythrophagocytosis rate, proteolytic activity, adhesion, and cytotoxic properties towards Caco2 colonic cancer cells were reduced in the presence of bLf. Lactoferrin could be a particularly important protein that naturally protects colonic epithelial cells from E. histolytica infection.