Human lactoferrin triggers a mitochondrial- and caspase-dependent regulated cell death in Saccharomyces cerevisiae.

We have previously shown that the antifungal activity of human lactoferrin (hLf) against Candida albicans relies on its ability to induce cell death associated with apoptotic markers. To gain a deeper understanding of the mechanisms underlying hLf-induced apoptosis, we characterized this cell death process in the well-established Saccharomyces cerevisiae model. Our results indicate that hLf induces cell death in S. cerevisiae in a manner that requires energy and de novo protein synthesis. Cell death is associated with nuclear chromatin condensation, preservation of plasma membrane integrity, and is Yca1p metacaspase-dependent. Lactoferrin also caused mitochondrial dysfunction associated with ROS accumulation and release of cytochrome c. Pre-incubation with oligomycin, an oxidative phosphorylation inhibitor, increased resistance to hLf and, accordingly, mutants deficient in the F1F0-ATP synthase complex were more resistant to death induced by hLf. This indicates that mitochondrial energetic metabolism plays a key role in the killing effect of hLf, though a direct role of F1F0-ATP synthase cannot be precluded. Overexpression of the anti-apoptotic protein Bcl-xL or pre-incubation with N-acetyl cysteine reduced the intracellular level of ROS and increased resistance to hLf, confirming a ROS-mediated mitochondrial cell death process. Mitochondrial involvement was further reinforced by the higher resistance of cells lacking mitochondrial DNA, or other known yeast mitochondrial apoptosis regulators, such as, Aif1p, Cyc3p and Aac1/2/3p. This study provides new insights into a detailed understanding at the molecular level of hLf-induced apoptosis, which may allow the design of new strategies to overcome the emergence of resistance of clinically relevant fungi to conventional antifungals.

Potassium efflux induced by a new lactoferrin-derived peptide mimicking the effect of native human lactoferrin on the bacterial cytoplasmic membrane.

A 31-amino acid synthetic peptide (NH(2)-FFSASCVPGADKGQFPNLCRLCAGTGENKCA-COOH) was chemically synthesized based on the amino acid sequence of a region of human lactoferrin homologous to other sequences present in the N- and C-lobes of all members of the transferrin family proteins. The peptide, termed kaliocin-1, and lactoferrin showed a bactericidal effect in assays performed in low-ionic-strength conditions. This is the first time that it is shown that the antimicrobial effect of lactoferrin depends on the extracellular cation concentration. The antimicrobial effect of kaliocin-1 was lower than that of human lactoferrin, but their activities were inhibited by Na(+) or K(+) in a cation concentration-dependent manner. In addition, the peptide was able to mimic native lactoferrin, inducing K(+)-efflux and a selective dissipation of the transmembrane electrical potential of Escherichia coli cells without causing extensive damage to the outer and inner bacterial membranes. In contrast, the peptide, but not lactoferrin, was able to permeabilize different ions through liposomal membranes. The hypothetical interaction of kaliocin-1 with a bacterial membrane compound is discussed based in the different ion flux induced on cellular and artificial membranes as well as data from circular dichroism assays. Kaliocin-1 was not cytotoxic and could be a suitable model for the design of analogs able to mimic the antibacterial effect of human lactoferrin.

Permeabilizing action of an antimicrobial lactoferricin-derived peptide on bacterial and artificial membranes.

A synthetic peptide (23 residues) that includes the antibacterial and lipopolysaccharide-binding regions of human lactoferricin, an antimicrobial sequence of lactoferrin, was used to study its action on cytoplasmic membrane of Escherichia coli 0111 and E. coli phospholipid vesicles. The peptide caused a depolarization of the bacterial cytoplasmic membrane, loss of the pH gradient, and a bactericidal effect on E. coli. Similarly, the binding of the peptide to liposomes dissipated previously created transmembrane electrical and pH gradients. The dramatic consequences of the transmembrane ion flux during the peptide exposure indicate that the adverse effect on bacterial cells occurs at the bacterial inner membrane.

Antimicrobial susceptibilities of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens spp. isolated in Spain.

The susceptibilities of 143 Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens isolates to 18 antimicrobial agents were tested. All P. gingivalis isolates were susceptible. In contrast, some Prevotella spp. (17%) were resistant to beta-lactams, erythromycin, clindamycin, or tetracycline and carried resistance genes, ermF or tetQ, or beta-lactamases.

Evaluation of the antimicrobial effect of lactoferrin on Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens.

The antimicrobial effect of lactoferrin (apoLf) on the oral, black-pigmented anaerobes Porphyromonas gingivalis, Prevotella intermedia and P. nitrescens has been studied. ApoLf did not kill any of these species but it did inhibit the growth of P. gingivalis, while iron-saturated Lf (FeLf) had no effect. The other two species were unaffected by apoLf. This growth inhibitory effect of apoLf could not be explained on the basis of chelation of inorganic iron, since growth of P. gingivalis occurred in the presence of ethylenediamine di-o-hydroxyphenylacetic acid provided haemin was added. Both apoLf and FeLf reduced haemin uptake by all three species and caused the release of cell-bound haemin in a dose-dependent manner. In addition, haemin reduced the binding of both apoLf and FeLf to P. intermedia and P. nigrescens but stimulated the binding of Lf by P. gingivalis. These data suggest that Lf forms complexes with haemin in solution and competes for the binding of haemin to certain cell receptors, possibly lipopolysaccharides, but this is not sufficient to inhibit growth of the bacteria. P. gingivalis appears to bind Lf-haemin complexes, as well as haemin alone, which may facilitate access of the Lf to the outer and cytoplasmic membranes of P. gingivalis, so disrupting function.

Endarteritis and mycotic aortic aneurysm caused by an oral strain of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans was isolated from blood cultures of a 33-year-old febrile patient with a previously undiagnosed coarctation of the aorta. Subgingival samples from diseased periodontal pockets revealed the presence of A. actinomycetemcomitans. An infected (mycotic) aortic aneurysm and endarteritis were diagnosed and surgically treated. The identity of blood and oral clinical isolates of A. actinomycetemcomitans was supported by genetic analysis, including fingerprinting by restriction fragment length polymorphism, ribotyping, and random amplified polymorphic DNA; biotyping; and antibiogram typing. These data strongly suggest that the periodontal pockets were the primary source of A. actinomycetemcomitans endarteritis in this case.

A beta-lactamase belonging to group 2e from oral clinical isolates of Prevotella intermedia.

A beta-lactamase in oral clinical isolates of Prevotella intermedia that hydrolyzed cefuroxime and cephalothin with rates of 600 and 53.3 respectively, relative to that for cephaloridine (100), was characterized as a 2e-cephalosporinase. Inhibition was observed by clavulanic acid (IC50 0.72 microM), tazobactam (IC50 0.21 microM) and sulbactam (IC50 0.07 microM) and was not inhibited by cloxacillin, EDTA, NaCl or p-CMB. The pI and pH optima were 4.7 and 5.4, respectively.

Identification of a lactoferrin-binding protein in Prevotella nigrescens.

A 40-kDa lactoferrin-binding protein was identified in a strain of Prevotella nigrescens isolated from a patient with periodontitis. The protein was purified by affinity column chromatography using a Sepharose-lactoferrin column and detergent-solubilized membranes. The N-terminal sequence revealed no apparent similarities with any other sequenced bacterial protein. The native conformation of the 40-kDa protein was a condition to bind either iron-free or iron-saturated lactoferrin. A possible function of this Lf-binding protein could be related with an iron acquisition mechanism in P. nigrescens.

Relationship between antibacterial activity and cell surface binding of lactoferrin in species of genus Micrococcus.

Human lactoferrin was bactericidal in vitro for Micrococcus luteus but not for other Micrococcus species (M. radiophilus, M. roseus and M. varians). A correlation between the binding of lactoferrin to the bacterial surface and the antimicrobial action was observed. Viability assays showed that ferric, but not ferrous, salts prevented binding and consequently M. luteus was not killed. The unsaturated form of lactoferrin showed a greater affinity than that of the iron-saturated molecule for lipomannan, a lipoglycan present on the cell wall of M. luteus, supporting the role for lipomannan as one of the possible binding sites of lactoferrin on M. luteus.

Binding and degradation of lactoferrin by Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens.

The ability of laboratory and clinical strains of Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens to bind and to degrade lactoferrin (Lf) has been assessed. Lf bound readily to whole cells of each species apparently via high-affinity site and one or more low-affinity sites. P. gingivalis showed a lower affinity for Lf than the other two species (P < 0.001). Virtually all strains of P. gingivalis completely degraded Lf under the conditions employed, whereas P. intermedia and P. nigrescens showed only partial degradation. These data suggest that Lf binds to a high-affinity receptor on all these bacteria and, particularly in the case of P. gingivalis, is then degraded by cell-associated proteases. This property may provide protection to the cell against the effects of Lf in periodontal sites and so is a possible virulence factor in disease. There was no association between the ability to degrade Lf and whether the strains had originated from healthy or diseased oral sites.

[Isolation of Haemophilus influenzae and Haemophilus parainfluenzae in genitourinary infections: a 4-year review]. / Aislamiento de Haemophilus influenzae y Haemophilus parainfluenzae en infecciones genitourinarias: una revisión de 4 años.

BACKGROUND: Haemophilus spp. had been previously suggested as a potential pathogen in genitourinary infections that could be sexually transmitted. In order to check that suggestions we have determined the incidence, pathogenic role, possible sexual transmission and susceptibility to antibiotics in isolates of Haemophilus parainfluenzae and Haemophilus influenzae from genital tract infections. The microbiological samples were taken during a period of four years from patients attended in a Service of Sexual Transmission Diseases and the data were further reviewed. METHODS: The study included 5,572 genital specimens from 2,182 women prostitutes with different genitourinary infections and from 825 men with urethritis. Microbiological samples were cultured in a non-specific media for genital pathogens and species of Haemophilus spp. and clinical circumstances of isolation were evaluated. Susceptibility tests were performed by using a standard microdilution test. RESULTS: Haemophilus spp. was isolated in 155 samples (2.8%) using a non-selective culture method. H. parainfluenzae was isolated in 100 cases (64.5%), Haemophilus influenzae in 45 cases (29%) and Haemophilus spp. in 10 strains (6.4%). Haemophilus spp. was isolated as a sole pathogen in men with urethritis (8 cases), epididymo-orchitis (2 cases), cervicitis and/or vaginitis (9 cases) and Bartholin's Abscess (2 cases). The most frequent biotypes were H. parainfluenzae biotype II (43%) and III (19%), and H. influenzae biotype IV (35.5%). Beta lactamase activity and ampicillin resistance were present in 29% of the H. parainfluenzae strains and in the 26.7% of clinical isolates of H. influenzae. CONCLUSIONS: 1) Haemophilus spp. was isolated from genitourinary infections at a low frequency in the studied group. 2) The pathogenic role of Haemophilus spp. was suggested when was isolated as a sole pathogen present from some infections of the genitourinary tract such as urethritis in men and Bartholin's abscess in women. 3) The susceptibility to antibiotics in the clinical isolates of Haemophilus spp. from genitourinary infections was similar previously reported studies performed in Spain.

Reliability of biologic indicators in a mail-return sterilization-monitoring service: a review of 3 years.

Most mail-return sterilization-monitoring services use spore strips to test sterilizers in dental clinics, but factors such as delay caused by mailing to the laboratory could cause false negatives. The aims of this study were to determine the influence of poststerilization time and temperature on the biologic indicator recovery system and to evaluate sterilization failure and its possible causes in dental clinics subscribing to a mail-return sterilization-monitoring service. Spore strips used in independent tests revealed the poststerilization time and temperature after a 7-day delay to have no significant influence. Sixty-six dental clinics that received quarterly biologic indicators to evaluate the effectiveness of their sterilizers had sterilization failure rates of 28.7% in 1992, 18.1% in 1993, and 9.1% in 1994, a statistically significant decrease in sterilization failure during the 3-year period. The usual causes of failure were operator error in wrapping of instruments, loading, operating temperature, or exposure time.

Interaction of calmodulin with lactoferrin.

Calmodulin, as a major intracellular calcium-binding protein, regulates many Ca(2+)-dependent enzymes and plays an important role in a wide spectrum of cellular functions of the eukaryotes. Interaction between calmodulin and human lactoferrin, a 78 kDa protein with antibacterial properties, was found in the presence of Ca2+ using (i) a method for the detection of calmodulin binding proteins with biotinylated calmodulin, (ii) affinity chromatography on an agarose-calmodulin column with subsequent detection by an enzyme-linked immunosorbent assay (ELISA). The binding of calmodulin to lactoferrin blocked the ability of lactoferrin to agglutinate Micrococcus lysodeikticus.

Phorbol myristate acetate-induced down-modulation of CD4 is dependent on calmodulin and intracellular calcium.

PMA causes rapid down-modulation of CD4 molecules on murine immature thymocytes, human PBL, and CD4-positive human tumor cell lines, but not on murine peripheral lymphocytes. The mechanisms of phorbol ester-induced down modulation of CD4 molecules, however, have not been elucidated. To determine how PMA down-modulates CD4 expression by T lymphocytes, we studied the ability of inhibitors of protein kinase C, calmodulin, actin, and tubulin to block PMA-induced modulation of CD4 in several murine and human cell types. We also tested the ability of intracellular and extracellular calcium chelators to block CD4 internalization. There was marked variability in the degree of PMA-induced down-modulation of CD4 among various cell types. The effects of PMA on CD4 expression were greater for murine thymocytes, for human PBL, and for the human lymphoblastic leukemia cell line, MOLT-3, than for any of the other cell types studied. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, blocked phosphorylation but not internalization of CD4 molecules induced by PMA. Therefore, phosphorylation of CD4 molecules by protein kinase C is not required for the internalization of the molecules. Internalization was blocked by both inhibitors of calmodulin, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide, and trifluoperazine. PMA-induced internalization of CD4 was blocked by Quin-2 AM, which chelates intracellular calcium. EGTA, which chelates extracellular calcium, did not block internalization. Inhibitors of actin or tubulin did not block internalization. These results suggest that PMA-induced modulation of CD4 can occur in the absence of phosphorylation of the CD4 molecules and is calmodulin and intracellular calcium dependent.

Calcium-dependent binding between calmodulin and lysozyme.

Calmodulin, an acidic protein that binds calcium with high affinity, has multiple roles in the activation of many enzymes involved in cellular regulation of eukaryotes. In this study we show that calmodulin binding to hen egg-white lysozyme, in a Ca2+-dependent way, was observed using electroblots incubated with biotinylated calmodulin and detected with avidin-alkaline phosphatase or for affinity chromatography on a gel calmodulin column. Antimicrobial activity of lysozyme was not modified in the presence of Ca2+-calmodulin.

Resistance to oleandomycin in Streptomyces antibioticus, the producer organism.

Resistance to oleandomycin in Streptomyces antibioticus, the producer organism, was studied. The organism was highly resistant in vivo to the antibiotic but sensitive to other macrolides and lincosamides. Protein synthesis in vivo by mycelium of S. antibioticus was more resistant to oleandomycin than that by mycelium of Streptomyces albus G, an oleandomycin-sensitive strain, and this resistance was dependent on the age of the culture, older mycelium of S. antibioticus being more resistant to oleandomycin than young mycelium. [3H]Oleandomycin was capable of binding to the same extent to the 50S subunits of the ribosomes of both organisms. Oleandomycin also inhibited in vitro protein synthesis by ribosomes obtained from an oleandomycin-production medium at the time when maximum levels of oleandomycin were being produced. A clear difference between the ability of the two organisms to incorporate exogenous oleandomycin was observed. Thus, while S. albus G took up oleandomycin, S. antibioticus showed a decreased permeability to the antibiotic, suggesting a role for cell permeability in self-resistance.