Decade-long use of the antimicrobial peptide combination tyrothricin does not pose a major risk of acquired resistance with gram-positive bacteria and Candida spp.

Tyrothricin, an antimicrobial peptide combination produced by Bacillus brevis consisting of gramicidins and tyrocidins commands broad antimicrobial activity against gram-positive bacteria and some yeasts in vitro. The polypeptide and its components have been used therapeutically for about 60 years in the local treatment of infected skin and infected oro-pharyngeal mucous membranes. Though older studies suggest that resistance development of originally susceptible microorganisms towards tyrothricin is a rare event, data concerning recent state of resistance are lacking. In the present in vitro study the susceptibility to tyrothricin of clinical isolates of bacterial and yeast origin from superficial swabs of the skin and mucous membranes of outpatients and inpatients obtained from clinical material in the second half of the year 2003 was determined. Using a microdilution assay, the minimum inhibitory concentration (MIC and MIC90, defined as the concentration that inhibits at least 90 percent of the tested strains) of 20 strains each of Staphylococcus aureus of the variety MSSA (susceptible to methicillin), Staphylococcus aureus of the variety MRSA (methicillin resistant), Staphylococcus haemolyticus, Streptococcus pyogenes, Enterococcus faecalis, Corynebacterium spec., Candida albicans and Candida parapsilosis was determined. All of the tested gram-positive bacteria turned out to be highly susceptible to tyrothricin with MICs ≤ 4mg/l. The tested yeast strains were susceptible to the polypeptide antibiotic as well, but (with MICs of 16 mg/l and 32 mg/l, respectively) to a lesser extent. No acquired resistance of the tested strains was determined, indicating that the risk of resistance development against topically applied tyrothricin is only marginal, if there is any at all. Thus, long-term-, i.e. decade-long use of topically applied tyrothricin and its components in the local treatment of infected skin does not pose a major risk with respect to acquired resistance of originally susceptible gram-positive bacteria and yeasts, not even in the case of Staphylococcus aureus, both with MSSA and MRSA strains. The broad anti-bacterial and anti-fungal activity of tyrothricin combined with its lacking risk for resistance development make the antimicrobial peptide a valuable addition to our therapeutic armamentarium in the treatment of infected skin.

Combined effect of liposomal and conventional amphotericin B in a mouse model of systemic infection with Candida albicans.

Amphotericin B may be employed as a potentially toxic deoxycholate complex or incorporated in liposomes and other particles which have an altered tissue distribution. A combination of both preparations could help to overcome the drawbacks of the individual preparations. We have employed a mouse model of systemic infection with Candida albicans to investigate whether this assumption was essentially true. We found that the combination of low dosages of both preparations (0.5 mg/kg of body weight/day of conventional and 0.5 mg kg of body weight/day liposomal amphotericin B) was more efficient than a similar dosage of conventional amphotericin B (1 mg/kg of body weight/day) in the liver and similar or higher dosages of liposomal amphotericin B (1 or 5 mg/kg of body weight/day) in the kidneys.

New molecular methods to study gene functions in Candida infections.

Candida albicans has become a model system for human pathogenic fungi in clinical research, mainly due to the increasing number of Candida infections. Molecular techniques to study C. albicans virulence properties have been improved over the last few years, despite difficulties in genetic manipulation of this fungus. Some of the recent achievements from our own laboratory or from other groups are described in this article. The molecular analysis of the recently identified ATP-dependent transporter Mlt1 using the green fluorescent protein (GFP) as reporter for protein localization and the dominant MPAR gene as a selection marker for gene inactivation provides an example for the study of gene functions in C. albicans.

Galactomannan enzyme immunoassay for monitoring systemic infection with Aspergillus fumigatus in mice.

Intravenous (i.v.) infection of immunocompetent mice with Aspergillus fumigatus was used to investigate the ability of a commercial galactomannan enzyme-linked immunosorbent assay (ELISA) to monitor the course of organ infection after dissemination. The test detected 100% of the fungemias which occurred for up to 5 days after infection. When blood-cultures became negative but there was a high load of fungi in the parenchymal organs and a positive culture from the brain, the ELISA was again positive in all animals. However, when blood cultures as well as brain cultures were negative and lower amounts of fungi demarcated by immune cells were present in the liver and kidneys which was the case between day 5 and 30 of infection, the test was negative in most of the animals. Therefore, the test was excellent for detection of early i.v. infection with Aspergillus fumigatus but not suited for detection of limited organ infection in immunocompetent mice.

Efficient treatment of murine systemic infection with Candida albicans using amphotericin B incorporated in nanosize range particles (emulsomes).

The effects of emulsome nanosize range lipid particles containing amphotericin B (EAmB) were compared with the reference formulation containing deoxycholate (Fungizone; Bristol-Myers Squibb, Munich, Germany) and with the commercial amphotericin lipid complex preparation (AmBisome; Nexstar, San Dimas, CA, USA). The minimal inhibitory concentrations of Fungizone and EAmB were identical although killing of Candida albicans was delayed when EAmB was used. In a tissue culture model and in mice, the incorporation of AmB into emulsomes resulted in a considerable reduction of toxicity in comparison with Fungizone. For comparison of the in vivo effect of the preparations a mouse model of systemic infection with C. albicans was used. All preparations were able to reduce the fungal burden in the liver and kidneys in comparison with control animals treated with isotonic saline. AmBisome was more efficient in the reduction of the fungal burden of the liver than EAmB and Fungizone, even when applied in a similar dosage of 1 mg kg(-1). In the kidneys, EAmB and Fungizone was slightly more effective than AmBisome. Therefore, in our models, the incorporation of AmB into nanosize particles was able to reduce toxicity without loss of efficiency. EAmB may be considered a candidate preparation for the treatment of infections with C. albicans in humans.

Systemic and intracerebral infections of mice with Listeria monocytogenes successfully treated with linezolid.

Linezolid is an oxazolidinone derivative which is active mostly against Gram-positive bacteria. In this work its activity against the facultatively intracellular bacterium Listeria monocytogenes was examined in vitro, in tissue culture and in animal models of systemic and intracerebral infection and compared with ampicillin which is the antibiotic of choice for treatment of listeriosis. All strains of L. monocytogenes were susceptible to the substance, with minimal inhibitory concentrations (MICs) determined by E-test ranging from 0.38 to 1.5 mg/l which is below the preliminary breakpoint of this substance. Linezolid was bacteriostatic against L. monocytogenes since up to 64 times the MIC did not kill the bacteria in 24 hours. Linezolid was also bacteriostatic on L monocytogenes in infected tissue culture cells. In animal models of systemic and intracerebral infection, linezolid was able to inhibit bacterial growth but was clearly less effective than ampicillin. In conclusion, linezolid might be useful for the treatment of infections with L monocytogenes in humans when ampicillin may not be used.

Influence of liposomal amphotericin B on CD8 T-cell function.

Liposomal amphotericin B was immunosuppressive on target cell lysis in vitro and on protection mediated by cytotoxic CD8 T cells in murine listeriosis. When dosages usually used for therapy in humans were compared, the immunosuppressive effect of 5 mg of liposomal amphotericin B/kg of body weight/day was similar to that of standard amphotericin B at 1 mg/kg/day, but a dosage of liposomal amphotericin B of 1 mg/kg/day was not suppressive in vivo.

Protein S-100B: a serum marker for ischemic and infectious injury of cerebral tissue.

The S-100B protein is released by injured astrocytes. After passage through a disintegrated blood-brain barrier (BBB) the molecule can be detected in the peripheral circulation. We investigated the association between the extent of brain injury and S-100B concentration in serum in cerebral injury caused by cerebral ischemia and cerebral fungal infection. Study I: The S-100B serum concentration was serially determined in 24 patients with ischemic stroke at 4, 8, 10, 24, 72 hours after the onset of symptoms. We observed that patients with brain lesions larger than 5 cm3 exhibited significantly increased serum levels of S-100B at 10, 24 and 72 hours compared to those with lesion volumes below 5 cm3. Furthermore, an association between S-100B serum concentration and neurological outcome was observed. Study II: In a mouse model of systemic fungal infection with Candida albicans we observed that serum levels of S-100B increased at day 1 after intravenous infection. At this time we could histologically demonstrate brain tissue injury by invading hyphae which had crossed the BBB. Furthermore, reactive astrogliosis was demonstrated by immunohistochemistry. On day 7 we found a significant decrease of S-100B serum level compared to day 1 and 4. This was associated with a demarcation of the fungi with leukocytes in brain tissue at this late phase of infection. No further invasion through the BBB was seen on day 7. In conclusion, serum levels of S-100B reflect the time course of tissue injury in cerebral ischemia and cerebral infection to a similar extent. Thus, S-100B may be a useful marker to assess cerebral tissue injury.

Crystal structure of the calcium-loaded spherulin 3a dimer sheds light on the evolution of the eye lens betagamma-crystallin domain fold.

BACKGROUND: The betagamma-crystallins belong to a superfamily of two-domain proteins found in vertebrate eye lenses, with distant relatives occurring in microorganisms. It has been considered that an eukaryotic stress protein, spherulin 3a, from the slime mold Physarum polycephalum shares a common one-domain ancestor with crystallins, similar to the one-domain 3-D structure determined by NMR. RESULTS: The X-ray structure of spherulin 3a shows it to be a tight homodimer, which is consistent with ultracentrifugation studies. The (two-motif) domain fold contains a pair of calcium binding sites very similar to those found in a two-domain prokaryotic betagamma-crystallin fold family member, Protein S. Domain pairing in the spherulin 3a dimer is two-fold symmetric, but quite different in character from the pseudo-two-fold pairing of domains in betagamma-crystallins. There is no evidence that the spherulin 3a single domain can fold independently of its partner domain, a feature that may be related to the absence of a tyrosine corner. CONCLUSION: Although it is accepted that the vertebrate two-domain betagamma-crystallins evolved from a common one-domain ancestor, the mycetezoan single-domain spherulin 3a, with its unique mode of domain pairing, is likely to be an evolutionary offshoot, perhaps from as far back as the one-motif ancestral stage. The spherulin 3a protomer stability appears to be dependent on domain pairing. Spherulin-like domain sequences that are found within bacterial proteins associated with virulence are likely to bind calcium.

Regulation of microglia by CD4+ and CD8+ T cells: selective analysis in CD45-congenic normal and Toxoplasma gondii-infected bone marrow chimeras.

Microglia, the resident macrophage population of the central nervous system, is rapidly activated in murine Toxoplasma encephalitis (TE). However, the precise contribution of microglia to intracerebral immune reactions and the in vivo regulation of microglial activity are still poorly understood. To selectively analyse microglial reactions in TE, we have established a model of radiation-induced CD45-congenic bone marrow chimeras between CD45.2+ C57BL/6 (recipient) and CD45.1+ B6.SJL (donor) mice. These chimeras allow a differentiation of radioresistant CD45.2+ microglia from all other leukocytes, which exhibit the CD45.1+ haplotype. In the normal brain, microglia produced tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-10, and IL-15 mRNA. In TE, marked microglial activation was observed with a de novo expression of IL-12p40 and inducible nitric oxide synthase mRNA, upregulation of IL-1beta and TNF-alpha mRNA, a continuous production of IL-10, and IL-15 mRNA, an induction of major histocompatibility class I and II antigens, intercellular adhesion molecule-1, and leukocyte function-associated antigen-1. Furthermore, selective depletion of CD4+ and/or CD8+ T cells in the chimeras revealed that microglial cytokine production was critically regulated by CD8+T cells, whereas expression of cell surface molecules was less dependent on T cells. These findings demonstrate a specific regulation of microglia by T lymphocytes during the course of TE.

Gamma S-crystallin of bovine and human eye lens: solution structure, stability and folding of the intact two-domain protein and its separate domains.

Human and bovine gammaS-crystallin (HgammaS and BgammaS) and their isolated N- and C-terminal domains were cloned and expressed as recombinant proteins in E. coli. HgammaS and BgammaS are found to be authentic according to their spectral and hydrodynamic properties. Both full-length proteins and isolated domains are monomeric and exhibit high thermal and pH stabilities. The thermodynamic characterization made use of chemically and thermally-induced equilibrium unfolding transitions at varying pH. In spite of its exemplary two-domain structure, gammaS-crystallin does not show bimodal unfolding characteristics. In the case of BgammaS, at pH 7.0, the C-terminal domain is less stable than the N-terminal one, whereas for HgammaS the opposite holds true. Differential scanning calorimetry confirms the results of chemically-induced equilibrium unfolding transitions. Over the whole pH range between 2.0 and 11.5, HgammaS-crystallin and its isolated domains (HgammaS-N and HgammaS-C) follow the two-state model. The two-state unfolding of the intact two-domain protein points to the close similarity of the stabilities of the constituent domains. Obviously, interactions between the domains do not contribute significantly to the overall stability of gammaS-crystallin. In contrast, the structurally closely related gammaB-crystallin owes much of its extreme stability to domain interactions.

Differential activation of a Candida albicans virulence gene family during infection.

The yeast Candida albicans is a harmless commensal in most healthy people, but it causes superficial as well as life-threatening systemic infections in immunocompromised patients. C. albicans can colonize or infect virtually all body sites because of its high adaptability to different host niches, which involves the activation of appropriate sets of genes in response to complex environmental signals. We have used an in vivo expression technology that is based on genetic recombination as a reporter of gene expression to monitor the differential activation of individual members of a gene family encoding secreted aspartic proteinases (Saps), which have been implicated in C. albicans virulence, at various stages of the infection process. Our results demonstrate that SAP expression depends on the type of infection, with different SAP isogenes being activated during systemic disease as compared with mucosal infection. In addition, the activation of individual SAP genes depends on the progress of the infection, some members of the gene family being induced immediately after contact with the host, whereas others are expressed only after dissemination into deep organs. In the latter case, the number of invading organisms determines whether induction of a virulence gene is necessary for successful infection. The in vivo expression technology allows the elucidation of gene expression patterns at different stages of the fungus-host interaction, thereby revealing regulatory adaptation mechanisms that make C. albicans the most successful fungal pathogen of humans and, at the same time, identifying the stage of an infection at which certain virulence genes may play a role.

Inefficient T cell memory in the brain of mice infected with Candida albicans.

We compared the contribution of T cell memory to the clearance of the fungus Candida albicans from the liver, kidneys and brain of Balb/c mice in a model of secondary systemic infection. In secondary infection, the fungi were more rapidly eliminated from the liver and kidneys than during primary infection. This was most pronounced in the liver where the fungi were eliminated at day 14 of infection. In contrast, in the brain, cultivable yeasts were still detectable 35 days after infection. Although both CD4(+) and CD8(+) cells could be detected in the brain with immunohistology, these cells appeared later in infection and in lower numbers than in the liver, and there were no significant differences in the numbers of T cells detected in the brain between primary and secondary infection. In contrast to the liver and the kidneys where an effect of T cells on the fungal load could be demonstrated, depletion of neither CD4(+) nor CD8(+) nor Thy-1.2(+) cells resulted in a significant increase of the amount of fungi in the brain above levels measured in secondarily infected mice treated with irrelevant antibodies. We conclude that the contribution of CD4(+) and CD8(+) cells to the clearance of C. albicans in secondary infection is organ-dependent and that T cell memory is inefficient in the brain.

Secreted lipases of Candida albicans: cloning, characterisation and expression analysis of a new gene family with at least ten members.

Extracellular lipolytic activity enabled the human pathogen Candida albicans to grow on lipids as the sole source of carbon. Nine new members of a lipase gene family (LIP2-LIP10) with high similarities to the recently cloned lipase gene LIP1 were cloned and characterised. The ORFs of all ten lipase genes are between 1281 and 1416 bp long and encode highly similar proteins with up to 80% identical amino acid sequences. Each deduced lipase sequence has conserved lipase motifs, four conserved cysteine residues, conserved putative N-glycosylation sites and similar hydrophobicity profiles. All LIP genes, except LIP7, also encode an N-terminal signal sequence. LIP3-LIP6 were expressed in all media and at all time points of growth tested as shown by Northern blot and RT-PCR analyses. LIP1, LIP3, LIP4, LIP5, LIP6 and LIP8 were expressed in medium with Tween 40 as a sole source of carbon. However, the same genes were also expressed in media without lipids. Two other genes, LIP2 and LIP9, were only expressed in media lacking lipids. Transcripts of most lipase genes were detected during the yeast-to-hyphal transition. Furthermore, LIP5, LIP6, LIP8 and LIP9 were found to be expressed during experimental infection of mice. These data indicate lipid-independent, highly flexible in vitro and in vivo expression of a large number of LIP genes, possibly reflecting broad lipolytic activity, which may contribute to the persistence and virulence of C. albicans in human tissue.

[Extracellular hydrolytic enzymes and their relevance during Candida albicans infections]. / Extrazelluläre hydrolytische Enzyme und ihre Relevanz bei Candida-albicans-Infektionen.

Candida albicans cannot only infect skin and mucosa, but can also cause life threatening systemic candidosis. While natural barriers and the immune system of healthy individuals normally prevent such infections, virulence factors exist that enable C. albicans to survive on surfaces and the permit the fungus to invade tissues and organs in immunocompromised patients. Adhesions factors, morphological flexibility and hydrolytic enzymes belong to this group of virulence factors.C.albicans appears to be able to use these specific virulence attributes at distinct stages of an infection or in different types of candidosis. For example, distinct adhension factors are important for the persistence of C. albicans on mucosal epithelial cells, while other factors are necessary for the adhesion to endothelial tissue. The differential expression of specific virulence factors at different stages of an infection could be the reason why C. albicans not only has single genes for extracellular hydrolytic enzymes, but gene families. Both secreted aspartate proteinases (Saps) and secreted lipases (Lips) from C. albicans are encoded by at least 10 different genes. This high number of similar genes might empower C. albicans with the ability to secrete a specific and appropriate enzymatic response at distinct stages of an infection. For both gene families differential expression has been shown in vitro and in vivo, which would be reasonable for such an adaptation. Expression studies revealed that distinct SAP and LIP genes were expressed under conditions when potential subtrates ( proteins or lipids) were not present in the growth medium. Such expression patterns would imply that these genes may have functions other than simply providing nutrients for the fungus. The specific transcription of single SAP genes during the course of an infection suggests that these genes may have specific functions during different stages of an infection. In fact, inhibition studies and the use of mutants with targeted gene disruptions showed that distinct SAP genes (SAP1-3) are important durning infections of skin and mucosa, while others (SAP4-6) are most relevant for systemic infections.

Germ tubes and proteinase activity contribute to virulence of Candida albicans in murine peritonitis.

Peritonitis with Candida albicans is an important complication of bowel perforation and continuous ambulatory peritoneal dialysis. To define potential virulence factors, we investigated 50 strains of C. albicans in a murine peritonitis model. There was considerable variation in their virulence in this model when virulence was measured as release of organ-specific enzymes into the plasma of infected mice. Alanine aminotransferase (ALT) and alpha-amylase (AM) were used as parameters for damage of the liver and pancreas, respectively. The activities of ALT and AM in the plasma correlated with invasion into the organs measured in histologic sections and the median germ tube length induced with serum in vitro. When the activity of proteinases was inhibited in vivo with pepstatin A, there was a significant reduction of ALT and AM activities. This indicates that proteinases contributed to virulence in this model. Using strains of C. albicans with disruption of secreted aspartyl proteinase gene SAP1, SAP2, SAP3, or SAP4 through SAP6 (collectively referred to as SAP4-6), we showed that only a Deltasap4-6 triple mutant induced a significantly reduced activity of ALT in comparison to the reference strain. In contrast to the Deltasap1, Deltasap2, and Deltasap3 mutants, the ALT induced by the Deltasap4-6 mutant could not be further reduced by pepstatin A treatment, which indicates that Sap4-6 may contribute to virulence in this model.

Stability of a homo-dimeric Ca(2+)-binding member of the beta gamma-crystallin superfamily: DSC measurements on spherulin 3a from Physarum polycephalum.

Spherulin 3a (S3a) from Physarum polycephalum represents the only known single-domain member of the superfamily of beta gamma eye-lens crystallins. It shares the typical two Greek-key motif and is stabilized by dimerization and Ca(2+)-binding. The temperature and denaturant-induced unfolding of S3a in the absence and in the presence of Ca2+ were investigated by differential scanning calorimetry and fluorescence spectroscopy. To accomplish reversibility without chemical modification of the protein during thermal denaturation, the only cysteine residue (Cys4) was substituted by serine; apart from that, the protein was destabilized by adding 0.5-1.8 M guanidinium chloride (GdmCl). The Cys4Ser mutant was found to be indistinguishable from natural S3a. The equilibrium unfolding transitions obey the two-state model according to N2-->2 U, allowing thermodynamic parameters to be determined by linear extrapolation to zero GdmCl concentration. The corresponding transition temperatures TM for the Ca(2+)-free and Ca(2+)-loaded protein were found to be 65 and 85 degrees C, the enthalpy changes delta Hcal, 800 and 1280 kJ/mol(dimer), respectively. The strong dependencies of TM and delta Hcal on the GdmCl concentration allow the molar heat capacity change delta Cp to be determined. As a result, delta Cp = 18 kJ/(K mol(dimer)) was calculated independent of Ca2+. No significant differences were obtained between the free energy delta G degree calculated from delta Hcal and TM, and extrapolated from the stability curves in the presence of different amounts of denaturant. The free energy derived from thermal unfolding was confirmed by the spectral results obtained from GdmCl-induced equilibrium transitions at different temperatures for the Ca(2+)-free or the Ca(2+)-loaded protein, respectively. Within the limits of error, the delta G degree values extrapolated from the transitions of chemical denaturation to zero denaturant concentration are identical with the calorimetric results.

Suppression of acquired immunity against Listeria monocytogenes by amphotericin B-mediated inhibition of CD8 T cell function.

Amphotericin B is frequently used for the treatment of fungal infections of immunocompromised individuals. Whereas immunomodulatory side effects of this agent are known, the influence of amphotericin B was studied in the model of murine Listeria monocytogenes infection. Treatment of L. monocytogenes-immune mice with a nontoxic dose of amphotericin B (0.75 mg/kg) reduced antilisterial protection by 4-5 orders of magnitude, while it had no significant effect on natural immunity against L. monocytogenes in naive mice. Treatment of mice with amphotericin B also abolished the protection mediated by transfer of an L. monocytogenes-specific CD8 T cell line. Furthermore, in vitro analysis showed that amphotericin B impaired target cell lysis and interferon-gamma production by peptide-specific CD8 T cell lines and antigen presentation by L. monocytogenes-infected macrophagelike cells. These data indicate that amphotericin B has a strong suppressive effect on the function of CD8 T cells in vitro and in vivo.

Kinetic and thermodynamic stabilization of the betagamma-crystallin homolog spherulin 3a from Physarum polycephalum by calcium binding.

Globular proteins may be stabilized, either intrinsically, at the various levels of the structural hierarchy, or extrinsically, by ligand binding. In the case of the dormant all-beta protein spherulin 3a (S3a) from the slime mold Physarum polycephalum, binding of calcium ions causes extreme kinetic and thermodynamic stabilization. S3a is the only known single-domain member of the two Greek key superfamily of betagamma-crystallins sharing the extreme long-term stability of its homologs in vertebrate eye lens. Spectral analysis allows two Ca2+-binding sites with KD=9 microM and 200 microM to be distinguished. Unfolding in the absence and in the presence of Ca2+gives evidence for extreme kinetic stabilization of the protein: In the absence of Ca2+, the half-time of unfolding in 2. 5 M guanidinium chloride (GdmCl) equals 8.3 minutes, whereas in the presence of Ca2+, even in 7.5 M GdmCl, it exceeds nine hours. To reach the equilibrium of unfolding in the absence and in the presence of Ca2+takes one day and eight weeks, respectively. The corresponding Gibbs free energies (based on the two-state model) are 77 and 135 kJ/mol. Saturation of S3a with Ca2+leads to an upward shift of the temperature-induced equilibrium transition by ca 20 deg. C. The in situ Ca2+concentration in the spherules is sufficient for the complete complexation of S3a in vivo.