muramidase and Klebsiella-Infections

This study examines the interaction of polymyxin B and colistin with the surface and outer membrane components of a susceptible and resistant strain of Klebsiella pneumoniae. The interaction between polymyxins and bacterial membrane and isolated LPS from paired wild type and polymyxin-resistant strains of K. pneumoniae were examined with N-phenyl-1-naphthylamine (NPN) uptake, fluorometric binding and thermal shift assays, lysozyme and deoxycholate sensitivity assays, and by (1)H NMR. LPS from the polymyxin-resistant strain displayed a reduced binding affinity for polymyxins B and colistin in comparison with the wild type LPS. The outer membrane NPN permeability of the resistant strain was greater compared with the susceptible strain. Polymyxin exposure enhanced the permeability of the outer membrane of the wild type strain to lysozyme and deoxycholate, whereas polymyxin concentrations up to 32 mg/ml failed to permeabilize the outer membrane of the resistant strain. Zeta potential measurements revealed that mid-logarithmic phase wild type cells exhibited a greater negative charge than the mid-logarithmic phase-resistant cells. Taken together, our findings suggest that the resistant derivative of K. pneumoniae can block the electrostatically driven first stage of polymyxin action, which thereby renders the hydrophobically driven second tier of polymyxin action on the outer membrane inconsequential.

Topics: 1-Naphthylamine; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Cell Membrane; Cell Membrane Permeability; Colistin; Deoxycholic Acid; Drug Interactions; Drug Resistance; Hydrophobic and Hydrophilic Interactions; Klebsiella Infections; Klebsiella pneumoniae; Lipopolysaccharides; Membrane Potentials; Microbial Sensitivity Tests; Muramidase; Polymyxin B; Protein Binding; Species Specificity; Static Electricity

Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in pulmonary host defense. Increased concentration of lysozyme in the airspaces of transgenic mice enhanced bacterial killing whereas lysozyme deficiency resulted in increased bacterial burden and morbidity. Lysozyme degrades peptidoglycan in the bacterial cell wall leading to rapid killing of Gram-positive organisms; however, this mechanism cannot account for the protective effect of lysozyme against Gram-negative bacteria. The current study was therefore designed to test the hypothesis that the catalytic activity (muramidase activity) of lysozyme is not required for bacterial killing in vivo. Substitution of serine for aspartic acid at position 53 (D53S) in mouse lysozyme M completely ablated muramidase activity. Muramidase-deficient recombinant lysozyme (LysM(D53S)) killed both Gram-positive and Gram-negative bacteria in vitro. Targeted expression of LysM(D53S) in the respiratory epithelium of wild-type (LysM(+/+)/LysM(D53S)) or lysozyme M(null) mice (LysM(-/-)/LysM(D53S)) resulted in significantly elevated lysozyme protein in the airspaces without any increase in muramidase activity. Intratracheal challenge of transgenic mice with Gram-positive or Gram-negative bacteria resulted in a significant increase in bacterial burden in LysM(-/-) mice that was completely reversed by targeted expression of LysM(D53S). These results indicate that the muramidase activity of lysozyme is not required for bacterial killing in vitro or in vivo.

Topics: Animals; Aspartic Acid; Blood Bactericidal Activity; Bronchoalveolar Lavage Fluid; Klebsiella Infections; Klebsiella pneumoniae; Mice; Mice, Knockout; Mice, Transgenic; Muramidase; Mutagenesis, Site-Directed; Peptidoglycan; Pseudomonas aeruginosa; Pseudomonas Infections; Recombinant Proteins; Respiratory Mucosa; Serine; Staphylococcal Infections; Staphylococcus aureus

Klebsiella pneumoniae is a common virulent causative agent for pneumonia. Lysozyme has previously been shown to play an important role in nonimmune host defense of the airways. This study was undertaken to assess the role of lysozyme M, the major isoform of lysozyme in mouse lung, in the killing of K. pneumoniae in lysozyme M(-/-) mice and transgenic mice with increased expression of lysozyme (lysozyme(tg) mice). The airways of lysozyme M(-/-) mice maintained in a pathogen-free facility were colonized by Lactobacilli, a component of the oropharyngeal flora. No lactobacilli were detected in the lungs of wild-type (WT) or lysozyme(tg) mice. Twenty-four hours after intratracheal infection with K. pneumoniae, bacterial killing was enhanced 9-fold in lysozyme(tg) mice compared with WT mice and 43-fold compared with lysozyme M(-/-) mice. In survival studies, no lysozyme M(-/-) mice survived beyond 72 hours after infection, whereas 75% of lysozyme(tg) (p < 0.01) and 25% of WT mice survived to 120 hours (p < 0.01). Deficiency of lysozyme M in the lungs increased susceptibility to K. pneumoniae infection, whereas increased expression of lysozyme conferred resistance to infection and enhanced survival.

Topics: Animals; Bronchoalveolar Lavage Fluid; Colony Count, Microbial; Cytokines; Klebsiella Infections; Klebsiella pneumoniae; Lung; Mice; Mice, Knockout; Mice, Transgenic; Muramidase; Respiratory Tract Infections; Survival Rate

Topics: Animals; Klebsiella Infections; Klebsiella pneumoniae; Lung; Mice; Muramidase; Respiratory Tract Infections

The constantly increasing number of substances with adjuvant activity outpaces the elucidation of their mode of action. This problem is of great importance as the immunomodulatory action of an adjuvant is time- and route-dependent, which implies that administration at a different moment or site may result in a reduced immune response. In the present work the possibility to achieve dual effect (stimulatory or inhibitory) is regarded in the light of the complement system. The object of the study is a preparation obtained by lysozyme digestion of Nocardia opaca cell walls (NLD). According to the results, the administration of NLD to mice (i.p. at a daily dose of 0.5 mg kg-1) during 3 days prior to the antigen resulted in an inhibition of serum antibody level against sheep red blood cells (SRBC) and lipopolysaccharide (LPS). At the same time, the preparation stimulated the antibody response to SRBC if it was applied after the antigen. The ability of NLD to ensure protection against experimental infection with Klebsiella pneumoniae was comparatively studied in complement-normal mice (strain ICR) and in C5-deficient mice (strain DBA/2). Firstly, it was established that complement-deficient mice were more resistant to infection than complement-normal. Secondly, the preparation expressed a protective effect in C5-deficient animals; nevertheless the inoculation was done s.c. or i.v. The departure of the infection depended on the rate of opsonization of K. pneumoniae. Under certain conditions NLD can provoke excessive C3 activation, which might aggravate the course of the infection. The preparation augmented the host response to second challenge with K. pneumoniae of complement-normal and C5-deficient mice.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Complement C5; Klebsiella Infections; Klebsiella pneumoniae; Male; Mice; Mice, Inbred DBA; Mice, Inbred ICR; Muramidase; Nocardia; Reference Values

Gram-negative bacterial pneumonias have been increasingly important as nosocomial infections. The following model was developed to study the pathogenesis and evaluate therapy of such infections. Intranasal instillation of rats with a suspension of 5 x 10(6) Klebsiella pneumoniae caused bronchopneumonia with 24 h. Bacteria were isolated from the lungs in large numbers (greater than 10(5) colony-forming units [CFU] for at least 13 days after inoculation. Thereafter, the viable concentration decreased to about 10(3) CFU at 21 days but increased to 10(4) CFU at 25 days. Mortality rarely exceeded 25%. Plasma zinc concentration decreased, and plasma seromucoid, lysozyme, and alpha2-macrofetoprotein increased during respiratory K. pneumoniae infection in rats. There seemed to be a linear relationship between seromucoid concentration and the concentration of K. pneumoniae in the lung expressed in log10 units. Plasma zinc, alpha2-macrofetoprtoein, or lysozyme levels, however, did not change until the concentration of bacteria retrieved fron lungs exceeded 4 to 5 logs, Analysis of blood samples obtained serially from the orbital sinuses revealed that rats that succumbed to infection had significantly higher levels of seromucoid, alpha2-macrofetoprotein, and lysozyme and lower levels of plasma zinc than infected rats that survived. Progressive increases in seromucoid and particularly in lysozyme and alpha2-macrofetoprotein appeared to be predicative of death. It is postulated that the threshold effect observed for alpha2-macrofetoprotein and lysozyme reflect significant damage to lung tissue, and thus these two variables are good indexes of the severity of this infection. We propose that this model may be of value in elucidating the pathogenesis of respiratory K. pneumoniae as well as in assessing various models of therapy.

Topics: alpha-Fetoproteins; Animals; Klebsiella Infections; Klebsiella pneumoniae; Lung; Male; Muramidase; Orosomucoid; Rats; Sepsis; Spleen; Zinc