lactoferrin and Pneumococcal-Infections

The pathogenicity of pneumococcus with high morbidity, mortality, and multi-drug resistance patterns has been increasing. The limited coverage of the licensed polysaccharide-based vaccines and the replacement of the non-vaccine serotypes are the main reasons for producing a successful serotype-independent vaccine. Pneumococcal surface protein A (PspA) is an extremely important virulence factor and an interesting candidate for conserved protein-based pneumococcal vaccine classified into two prominent families containing five clades. PspA family-elicited immunity is clade-dependent, and the level of the PspA cross-reactivity is restricted to the same family.. To cover and overcome the clade-dependent immunity of the PspAs in this study, we designed and tested a PspA. For the first time, this work suggested a novel PspA-based vaccine candidate using immunoinformatics tools. The designed PspA. Our findings elucidated the potential application of the PspA

Topics: Animals; Antibodies; Antibodies, Bacterial; Bacterial Proteins; Epitopes; Humans; Lactoferrin; Mice; Mice, Inbred BALB C; Molecular Docking Simulation; Pneumococcal Infections; Pneumococcal Vaccines; Serogroup; Streptococcus pneumoniae

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.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacterial Proteins; Carbon-Sulfur Lyases; Cattle; Child; Gene Expression; Genes, Bacterial; Humans; Lactoferrin; Peptide Fragments; Pneumococcal Infections; Recombinant Fusion Proteins; Streptococcus pneumoniae

Targeting an antigen to Fc receptors (FcR) can enhance the immune response to the antigen in the absence of adjuvant. Furthermore, we recently demonstrated that intranasal immunization with an FcγR-targeted antigen enhances protection against a category A intracellular mucosal pathogen, Francisella tularensis. To determine if a similar strategy could be applied to the important pathogen Streptococcus pneumoniae, we used an improved mucosal FcR-targeting strategy that specifically targets human FcγR type I (hFcγRI). A humanized single-chain antibody component in which the variable domain binds to hFcγRI [anti-hFcγRI (H22)] was linked in a fusion protein with the pneumococcal surface protein A (PspA). PspA is known to elicit protection against pneumococcal sepsis, carriage, and pneumonia in mouse models when administered with adjuvants. Anti-hFcγRI-PspA or recombinant PspA (rPspA) alone was used to intranasally immunize wild-type (WT) and hFcγRI transgenic (Tg) mice in the absence of adjuvant. The hFcγRI Tg mice receiving anti-hFcγRI-PspA exhibited elevated S. pneumoniae-specific IgA, IgG2c, and IgG1 antibodies in serum and bronchoalveolar lavage fluid. Neither immunogen was effective in protecting WT mice in the absence of adjuvant, but when PspA was targeted to hFcγRI as the anti-hFcγRI-PspA fusion, enhanced protection against lethal S. pneumoniae challenge was observed in the hFcγRI Tg mice compared to mice given nontargeted rPspA alone. Immune sera from the anti-hFcγRI-PspA-immunized Tg mice showed enhanced complement C3 deposition on bacterial surfaces, and protection was dependent upon an active complement system. Immune serum also showed an enhanced bactericidal activity directed against S. pneumoniae that appears to be lactoferrin mediated.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animals; Antibodies, Bacterial; Bacterial Proteins; Blood Bactericidal Activity; Bronchoalveolar Lavage Fluid; Complement System Proteins; Humans; Immunoglobulin A; Immunoglobulin G; Lactoferrin; Mice; Mice, Inbred C57BL; Pneumococcal Infections; Pneumococcal Vaccines; Receptors, Fc; Recombinant Fusion Proteins; Serum; Single-Chain Antibodies; Streptococcus pneumoniae; Survival Analysis; Vaccines, Synthetic

It is known that apolactoferrin, the iron-free form of human lactoferrin, can kill many species of bacteria, including Streptococcus pneumoniae. Lactoferricin, an N-terminal peptide of apolactoferrin, and fragments of it are even more bactericidal than apolactoferrin. In this study we found that apolactoferrin must be cleaved by a serine protease in order for it to kill pneumococci. The serine protease inhibitors were able to block killing by apolactoferrin but did not block killing by a lactoferrin-derived peptide. Thus, the killing of pneumococci by apolactoferrin appears to require a protease to release a lactoferricin-like peptide(s). Incubation of apolactoferrin with growing pneumococci resulted in a 12-kDa reduction in its molecular mass, of which about 7 to 8 kDa of the reduction was protease dependent. Capsular type 2 and 19F strains with mutations in the gene encoding the major cell wall-associated serine protease, prtA, lost much of their ability to degrade apolactoferrin and were relatively resistant to killing by apolactoferrin (P < 0.001). Recombinant PrtA was also able to cleave apolactoferrin, reducing its mass by about 8 kDa, and greatly enhance the killing activity of the solution containing the apolactoferrin and its cleavage products. Mass spectroscopy revealed that PrtA makes a major cut between amino acids 78 and 79 of human lactoferrin, removing the N-terminal end of the molecule (about 8.6 kDa). The simplest interpretation of these data is that the mechanism by which apolactoferrin kills Streptococcus pneumoniae requires the release of a lactoferricin-like peptide(s) and that it is this peptide(s), and not the intact apolactoferrin, which kills pneumococci.

Topics: Apoproteins; Blotting, Western; Cloning, Molecular; Host-Pathogen Interactions; Humans; Lactoferrin; Pneumococcal Infections; Recombinant Proteins; Serine Proteases; Serine Proteinase Inhibitors; Streptococcus pneumoniae; Tandem Mass Spectrometry

To find the effect of apolactoferrin administration on the middle and inner ears after experimentally induced pneumococcal otitis media.. Histopathologic and morphometric analysis of the middle and inner ears.. University of Minnesota, Minneapolis.. Ten chinchillas.. The middle ear cavities of chinchillas were inoculated bilaterally with type 2 wild-type Streptococcus pneumoniae. Twenty-four hours later, the ears of 5 of the animals were injected with phosphate-buffered saline (PBS) and the other 5 with human apolactoferrin. The animals were killed 24 hours after the last injection. Bacterial counts were made of the middle ear effusions, and the cochleae were processed for histologic analysis. The thickness of the round window membranes and bacterial and inflammatory cell infiltration of the round window membranes, and scala tympani and damage of the hair cells and stria vascularis were compared for these 2 groups of animals.. Comparison of inflammatory and bacterial cells in the middle and inner ears, and damage to inner ear structures.. Bacterial plate counts of middle ear effusions (P  = .005) and the number of inflammatory cells in the round window membrane (P  = .047) were significantly lower in the apolactoferrin group compared with the group treated with PBS.. Further investigation of apolactoferrin as a nonantibiotic approach for the treatment of otitis media and its complications is needed to confirm its safety and efficacy.

Topics: Animals; Apoproteins; Chinchilla; Lactoferrin; Otitis Media; Pneumococcal Infections; Streptococcus pneumoniae

Pneumococcal surface protein A (PspA), a virulence factor of Streptococcus pneumoniae, is exceptionally diverse, being classified into two major families which are over 50% divergent by sequence analysis. A family 1 PspA from strain WU2 was previously shown to impede the clearance of pneumococci from mouse blood and to interfere with complement deposition on the bacterial surface. To determine whether a family 2 PspA can perform the same role as family 1 PspA, the family 1 PspA (from strain WU2) was replaced with a family 2 PspA (from strain TIGR4) by molecular genetic methods to make an isogenic pair of strains expressing different PspA proteins. Surface binding of lactoferrin and interference with C3 deposition by the two types of PspA proteins were determined by flow cytometry, and virulence was assessed in a mouse bacteremia model. Although the family 2 PspA appeared to bind less human lactoferrin than did the family 1 PspA, both PspA proteins could interfere with complement deposition on the pneumococcal surface and could provide full virulence in the mouse infection model. A mutant form of the family 2 PspA with a deletion within the choline-binding region was also produced. Pneumococci with this mutant PspA failed to bind human lactoferrin even though the PspA was present on the pneumococcal surface. The mutant PspA only partially interfered with complement deposition and moderately attenuated virulence. These results suggest that family 1 and family 2 PspA proteins play similar roles in virulence and that surface accessibility of PspA is important for their function.

Topics: Amino Acid Sequence; Animals; Antigens, Bacterial; Antigens, Surface; Bacteremia; Bacterial Proteins; Complement Activation; Complement C3; Disease Models, Animal; Female; Humans; Lactoferrin; Mice; Mice, Inbred CBA; Molecular Sequence Data; Mutation; Pneumococcal Infections; Sequence Analysis, DNA; Streptococcus pneumoniae; Virulence

The changes in intraneutrophilic and plasma concentrations of the three antibacterial proteins lysozyme, lactoferrin, and myeloperoxidase were studied sequentially during acute bacterial infection in nine patients. Intraneutrophilic concentrations of the three proteins were decreased by more than 50% during the 1st week of infection, followed by a slow increase over the following 2 weeks. Nadir values coincided with maximal toxic granulation of the neutrophils. The data suggest that neutrophilic granulocytes are deficient during early bacterial infection, possibly because of deficient synthesis of antibacterial proteins in the bone marrow, and that neutrophil toxic granulation is the visual counterpart of this defect. The plasma concentrations of the three proteins showed considerable differences: whereas plasma lysozyme did not show any sequential changes, plasma myeloperoxidase was high at the start of infection and quickly decreased towards normal values, and plasma lactoferrin, high in the first samples, showed a secondary peak 1 week after onset of disease, before normalization was seen. These differences may result from differences in the signals are specific for the individual antibacterial protein and not for the different types of neutrophil granules.

Topics: Adult; Aged; Bacterial Infections; Blood Bactericidal Activity; Humans; Lactoferrin; Lactoglobulins; Meningitis; Middle Aged; Muramidase; Neutrophils; Peroxidase; Peroxidases; Pneumococcal Infections; Time Factors