Direct observation of Staphylococcus aureus cell wall digestion by lysostaphin

The advent of Staphylococcus aureus strains that are resistant to virtually all antibiotics has increased the need for new antistaphylococcal agents. An example of such a potential therapeutic is lysostaphin, an enzyme that specifically cleaves the S. aureus peptidoglycan, thereby lysing the bacteria. Here we tracked over time the structural and physical dynamics of single S. aureus cells exposed to lysostaphin, using atomic force microscopy. Topographic images of native cells revealed a smooth surface morphology decorated with concentric rings attributed to newly formed peptidoglycan. Time-lapse images collected following addition of lysostaphin revealed major structural changes in the form of cell swelling, splitting of the septum, and creation of nanoscale perforations. Notably, treatment of the cells with lysostaphin was also found to decrease the bacterial spring constant and the cell wall stiffness, demonstrating that structural changes were correlated with major differences in cell wall nanomechanical properties. We interpret these modifications as resulting from the digestion of peptidoglycan by lysostaphin, eventually leading to the formation of osmotically fragile cells. This study provides new insight into the lytic activity of lysostaphin and offers promising prospects for the study of new antistaphylococcal agents.     

Induction of Prophages by Fluoroquinolones in Streptococcus pneumoniae: Implications for Emergence of Resistance in Genetically-Related Clones

Antibiotic resistance in Streptococcus pneumoniae has increased worldwide by the spread of a few clones. Fluoroquinolone resistance occurs mainly by alteration of their intracellular targets, the type II DNA topoisomerases, which is acquired either by point mutation or by recombination. Increase in fluoroquinolone-resistance may depend on the balance between antibiotic consumption and the cost that resistance imposes to bacterial fitness. In addition, pneumococcal prophages could play an important role. Prophage induction by fluoroquinolones was confirmed in 4 clinical isolates by using Southern blot hybridization. Clinical isolates (105 fluoroquinolone-resistant and 160 fluoroquinolone-susceptible) were tested for lysogeny by using a PCR assay and functional prophage carriage was studied by mitomycin C induction. Fluoroquinolone-resistant strains harbored fewer inducible prophages (17/43) than fluoroquinolone-susceptible strains (49/70) (P = 0.0018). In addition, isolates of clones associated with fluoroquinolone resistance [CC156 (3/25); CC63 (2/20), and CC81 (1/19)], had lower frequency of functional prophages than isolates of clones with low incidence of fluoroquinolone resistance [CC30 (4/21), CC230 (5/20), CC62 (9/21), and CC180 (21/30)]. Likewise, persistent strains from patients with chronic respiratory diseases subjected to fluoroquinolone treatment had a low frequency of inducible prophages (1/11). Development of ciprofloxacin resistance was tested with two isogenic strains, one lysogenic and the other non-lysogenic: emergence of resistance was only observed in the non-lysogenic strain. These results are compatible with the lysis of lysogenic isolates receiving fluoroquinolones before the development of resistance and explain the inverse relation between presence of inducible prophages and fluoroquinolone-resistance.     

A new D-2-hydroxyacid dehydrogenase with dual coenzyme-specificity from Haloferax mediterranei, sequence analysis and heterologous overexpression

A gene encoding a new D-2-hydroxyacid dehydrogenase (E.C. 1.1.1.) from the halophilic Archaeon Haloferax mediterranei has been sequenced, cloned and expressed in Escherichia coli cells with the inducible expression plasmid pET3a. The nucleotide sequence analysis showed an open reading frame of 927 bp which encodes a 308 amino acid protein. Multiple amino acid sequence alignments of the D-2-hydroxyacid dehydrogenase from H. mediterranei showed high homology with D-2-hydroxyacid dehydrogenases from different organisms and other enzymes of this family. Analysis of the amino acid sequence showed catalytic residues conserved in hydroxyacid dehydrogenases with d-stereospecificity. In the reductive reaction, the enzyme showed broad substrate specificity, although alpha-ketoisoleucine was the most favourable of all alpha-ketocarboxylic acids tested. Kinetic data revealed that this new D-2-hydroxyacid dehydrogenase from H. mediterranei exhibits dual coenzyme-specificity, using both NADPH and NADH as coenzymes. To date, all D-2-hydroxyacid dehydrogenases have been found to be NADH-dependent. Here, we report the first example of a D-2-hydroxyacid dehydrogenase with dual coenzyme-specificity.     

Pathogenicity ofPseudomonas aeruginosa and its relationship to the choline metabolism through the action of cholinesterase,acid phosphatase,and phospholipase C

The increase of cholinesterase (ChE), acid phosphatase (Ac.Pase), and phospholipase C (PLC) activities byPseudomonas aeruginosa was associated with the choline consumption in growth media of varied composition (high or low P_i concentrations, presence or absence of ammonium ion, amino acids, polyamines, peptone, or tricarboxylic acid cycle intermediates). The highest production of the three enzymes occurred in the late stationary growth phase. The simultaneous presence of alkaline phosphatase (Alk.Pase) and the above enzymes was noted when the bacteria were grown in low P_i medium plus choline, in the absence of a preferred carbon source. The importance of choline in the production of ChE, Ac.Pase, and PLC was observed in either clinical isolates or collection strains ofP. aeruginosa. These enzymes catalyze the hydrolysis of acetylcholine, phosphorylcholine, and phosphatidylcholine. Through their action the bacteria may break down various compounds (e.g., acetylcholine, from the corneal epithelium; lung surfactant dipalmitoylphosphatidylcholine; phosphorylcholine, a product of the PLC action) or cell membranes through the coordinated action of PLC and Ac.Pase or Alk.Pase. The final consequence of the action of these enzymes is an increase of the free choline concentration. Extrapolated to an in vivo situation, if the stationary growth phase resembles the conditions thatP. aeruginosa encounters in its natural environments, then it is possible to include choline among the factors promoting the pathogenicity of this bacterium.     

Determination of the reactivities and cross-reactivities of monoclonal antibodies against staphylococcal enterotoxin A by indirect ELISA and immunoblot including a semiautomated electrophoresis system

Eight murine monoclonal antibodies (Mabs) to staphylococcal enterotoxin A (SEA) were produced using standard hybridoma techniques. We studied reactivities and cross-reactivities by indirect ELISA and immunoblotting. Two of these Mabs (A5 and A7) reacted with five serovars (A-E) of SE in both systems. Only Mab A1 reacted specifically with the homologous toxin, while four Mabs reacted with SEA and SEE. Mabs A5 and A7 could be used to detect all five serovars of SEs in a single assay.     

Carnitine resembles choline in the induction of cholinesterase,acid phosphatase,and phospholipase C and in its action as an osmoprotectant in Pseudomonas aeruginosa

The present study demonstrates that under conditions of iso or hyperosmolarity, P. aeruginosa utilized carnitine as the carbon, nitrogen or carbon and nitrogen sources. As occurred in the case of choline, the bacteria synthesized cholinesterase (ChE), acid phosphatase (Ac.Pase) and phospholipase C (PLC) under any of these conditions and in the presence of high or low P_i concentrations.Carnitine acted as an osmoprotectant when the cells were grown in the presence of preferred carbon and nitrogen sources and high NaCl concentrations. Under these conditions the three enzyme activities were not produced.The osmotically stressed bacteria grown under any of the above conditions accumulated betaine. Its presence indicated that carnitine may be metabolized by P. aeruginosa to produce betaine which could account for the induction of the three enzyme activities or its action as an osmoprotectant.The phosphatidylcholine encountered in the host cell membranes allows the bacteria to obtain free choline by the coordinated action of PLC and Ac.Pase. Since the consequence of this action may be cell disruption, the increase of free carnitine in the natural environment of the bacteria is also possible. These two compounds, choline and carnitine, acting in conjunction or separately, may increase the production of PLC and Ac.Pase activities by P. aeruginosa and thus enhance the degradative effect upon the host cells.     

Structural identity between HLA-A2 antigens differentially recognized by alloreactive cytotoxic T lymphocytes

Alloreactive CTL raised against HLA-A2 Ag often display heterogeneous recognition of HLA-A2+ target cells. This heterogeneity has been found to reflect structural polymorphism among the corresponding target Ag, thus defining HLA-A2 subtypes. A previous study (van der Poel et al. 1986. Human Immunol. 16:247) established the existence of a new HLA-A2.4 variant, A2-SCHU, that was distinguished from A*0206 (A2.4a) by HLA-A2-specific alloreactive CTL. The same CTL subdivided HLA-A2.1 Ag into two subgroups. In the present study, the molecular basis of this heterogeneity has been examined by double-label comparative peptide mapping analysis of differentially recognized A2.1 and A2.4 Ag. In addition, we have determined the complete sequence of polymerase chain reaction-amplified full length cDNA from A2-SCHU. The results show that: 1) A2-SCHU is indistinguishable from A*0206 by peptide mapping; 2) the cDNA sequence of A2-SCHU is identical to that of A*0206; and 3) two differentially recognized A2.1 Ag are both indistinguishable from A*0201 by comparative peptide mapping. These results indicate that differential recognition by alloreactive CTL can occur among structurally identical class I HLA Ag and suggest that allorecognition by such CTL may involve corecognition of endogenous peptides, presumably derived from polymorphic proteins.     

Pseudomonas aeruginosa acid phosphatase contains an anionic site with a trimethyl subsite

Summary In this work the action of the following compounds upon Ps. aeruginosa acid phosphatase has been studied: 1) alkylammonium compounds; 2) aminoalcohols and aminoacids with different substituents (–H, –CH₃OH and –CH₃) attached to the nitrogen atom; 3) alcohols analogous to some compounds of the above series, but without the amino group.It was found that the enzyme inhibition was more effective with N-trimethylated compounds than with the triethylated ones. The degree of inhibition depended on the number of methyl groups bound to the nitrogen atom. Taking into account the choline and betaine series the hydroxyl derivatives showed more affinity for the enzyme than the carboxylated ones. In each series the Ki values increased with the decrease of methyl groups bound to the nitrogen atom. The presence of a positively charged nitrogen atom in the molecule of the effector was essential. These results enable us to confirm that in the molecule of Ps. aeruginosa acid phosphatase there exists an anionic site with one subsite with affinity for methyl groups.     

Choline and betaine as inducer agents of Pseudomonas aeruginosa phospholipase C activity in high phosphate medium

In Pseudomonas aeruginosa, choline or betaine employed as the sole carbon and nitrogen source in a high phosphate medium induced a phospholipase C and an acid phosphatase activity but not an alkaline phosphatase activity. The P. aeruginosa strain utilized in this work does not possess a constitutive phospholipase C, since under culture conditions identical to those utilized by other authors (J. Bacteriol. 93, 670-674 (1967) and J. Bacteriol. 150, 730-738 (1982), our phospholipase C proved to be an inorganic phosphate-repressible enzyme. These findings enable us to conclude that although the phosphate control for the synthesis of phospholipase C may exist, it is expressed only under certain favorable culture conditions.     

Phosphodiesterase 7A1 is expressed in human CD4+ naïve T cells at higher levels than in CD4+ memory cells and is not required during their CD3/CD28-dependent activation

PDE7A1 is a cAMP-hydrolyzing phosphodiesterase expressed in lymphoid tissue, where its possible role during T cell activation remains unclear. We have characterized the functional relevance of PDE7A1 in the na?ve (CD4+CD45RA+) and memory (CD4+CD45RO+) subsets of human peripheral CD4+ T cells during CD3/CD28-dependent stimulation. Our results indicate that PDE7A1 is expressed in resting na?ve CD4+ T cells at higher levels than in the corresponding memory cells and that levels of PDE7A1 mRNA are not upregulated upon CD3/CD28 mediated stimulation of these T cell subsets. Treatment with a selective inhibitor of PDE7A1 does not impair CD3/CD28 induced activation of na?ve or memory CD4+ T cells, nor does it increase intracellular cAMP in CD4+ T cells. We conclude that PDE7A1 is not required during CD3/CD28-dependent activation of na?ve and memory CD4+ T cells, but cannot rule out other regulatory roles of PDE7A1 during maturation of CD4+ T cells.