A plasmid-encoded nicotinamidase (PncA) is essential for infectivity of Borrelia burgdorferi in a mammalian host

Borrelia burgdorferi, a spirochaete that causes Lyme borreliosis, contains 21 linear and circular plasmids thought to be important for survival in mammals or ticks. Our results demonstrate that the gene BBE22 encoding a nicotinamidase is capable of replacing the requirement for the 25 kb linear plasmid lp25 during mammalian infection. Transformation of B. burgdorferi lacking lp25 with a shuttle vector containing the lp25 gene BBE22 (pBBE22) restored infectivity in mice to a level comparable to that of wild-type Borrelia. This complementation also restored the growth and host adaptation of lp25-B. burgdorferi in dialysis membrane chambers (DMCs) implanted in rats. A single Cys to Ala conversion at the putative active site of BBE22 abrogated the ability of pBBE22 to re-establish infectivity or growth in DMCs. Additional Salmonella typhimurium complementation studies and enzymatic analysis demonstrated that the BBE22 gene product has nicotinamidase activity and is most probably required for the biosynthesis of NAD. These results indicate that some plasmid-encoded products fulfil physiological functions required in the enzootic cycle of pathogenic Borrelia.     

Antibiotic production by strains of Gliocladium virens and its relation to the biocontrol of cotton seedling diseases

Strains of the fungal antagonist Gliocladium virens were separated into two distinct groups on the basis of secondary metabolite production in vitro. Strains of the ‘P’ group produced the antibiotics gliovirin and heptelidic acid but not the antibiotic gliotoxin and its companion, dimethylgliotoxin. Strains of the ‘Q’ group produced gliotoxin and dimethylgliotoxin but not gliovirin or heptelidic acid. Strains from both groups produced the antibiotic viridin and phytotoxin viridiol. Gliovirin was very inhibitory to Pythium ultimum but had no activity against Rhizoctonia solani, and strains that produce it were more effective seed treatment biocontrol agents of disease incited by P. ultimum. Conversely, gliotoxin was more active against R. solani than against P. ultimum, and strains that produced it were more effective seed treatments for controlling disease incited by R. solani. These results indicate that the antibiotic profiles of strains should be considered when screening strains for biocontrol efficacy, and that it may be necessary to treat seeds with a combination of strains in order to broaden the disease control spectrum.     

Circular dichroic, infrared and other studies on the protein component of pig brain thromboplastin.

1. A four-step procedure used to isolate the protein component (apoprotein III) of pig brain thromboplastin yielded approximately 25 mg from 500g of brain. 2. In the absence of detergent, apoprotein III had an apparent mol.wt. of 360 000 by gel-filtration, and, after electrophoresis on polyacrylamide gels in the presence of sodium docecyl sulphate, it appeared as a major protein band of mol.wt.59 000, suggesting the existence of polymeric and monomeric forms. 3. Chemical analyses of apoprotein III revealed that hydrophilic and hydrophobic amino acids were present in a ratio of 3:2, together with approx, 9% (w/w) of carbohydrate. 4. The far-u.v.c.d. and i.r. spectral data indicated that, like other membrane proteins, apoprotein III has a high percentage of unordered structure with lesser amounts of alpha and beta-forms. 5. Relipidation of apoprotein III to restore clotting activity caused no extensive alteration in the c.d. and i.r. spectra, indicating that the phospholipid associates with a comparatively small hydrophobic segment. The constrained unordered conformation, which makes the major contribution to the c.d. spectrum, probably forms a separate domain in the aqueous phase. The absence of any increase in the amplitude of both negative c.d. extrema, following relipidation, contrasted with the substantial increase observed in a helix-forming solvent and raises the possibility that the more stable polymeric form of apoprotein III is retained as the active form in the lipid phase. 6. We suggest that as a consequence of cell membrane damage, the recognition and activation of factor VII may involve minor changes of conformation that are dependent upon the flexibility inherent in an unordered secondary structure.     

Substrate induced conformational changes in argininosuccinate synthetase.

Argininosuccinate synthetase (AS) is the rate-limiting enzyme of both the urea and arginine-citrulline cycles. In mammals, deficiency of AS leads to citrullinemia, a debilitating and often fatal autosomal recessive urea cycle disorder, whereas its overexpression for sustained nitric oxide production via the arginine-citrulline cycle leads to the potentially fatal hypotension associated with septic and cytokine-induced circulatory shock. The crystal structures of Escherichia coli argininosuccinate synthetase (EAS) in complex with ATP and with ATP and citrulline have been determined at 2.0-A resolution. These are the first EAS structures to be solved in the presence of a nucleotide substrate and clearly identify the residues that interact with both ATP and citrulline. Two distinct conformations are revealed for ATP, both of which are believed to be catalytically relevant. In addition, comparisons of these EAS structures with those of the apoenzyme and EAS complexed with aspartate and citrulline (Lemke, C. T., and Howell, P. L. (2001) Structure (Lond.) 9, 1153-1164) provide structural evidence of ATP-induced conformational changes in the nucleotide binding domain. Combined, these structures also provide structural explanations of some of the observed kinetic properties of the enzyme and have enabled a detailed enzymatic mechanism of AS catalysis to be proposed.     

Effects of repetitive activity, ruthenium red, and elevated extracellular calcium on frog skeletal muscle: implications for t-tubule conduction

In this report we review evidence that indicates that experimental elevation of t-tubular calcium can lead to failure of action potential propagation within the t system and we present some new evidence suggesting that t-tubular calcium concentration may rise during repetitive activity. The evidence for t-tubular conduction failure consists of comparisons of the effects of high calcium and of ruthenium red on excitation and excitation-contraction coupling as well as morphological observations of wavy myofibrils in the axial core of fibers contracting tetanically in solutions containing elevated calcium concentrations. Evidence for elevation of t-tubular calcium concentration during repetitive activity comes from the following. During twitches, the early, large birefringence signal and force development are delayed in onset if the extracellular calcium and (or) potassium concentrations are above normal or if the fiber has been stimulated tetanically just prior to the test twitch. The delays that occur in twitches following tetanic contractions are attenuated when the extracellular and, therefore, the t-tubular calcium concentration is buffered with citrate.     

Autocatalytic activation of a malarial egress protease is druggable and requires a protein cofactor

Malaria parasite egress from host erythrocytes (RBCs) is regulated by discharge of a parasite serine protease called SUB1 into the parasitophorous vacuole (PV). There, SUB1 activates a PV‐resident cysteine protease called SERA6, enabling host RBC rupture through SERA6‐mediated degradation of the RBC cytoskeleton protein β‐spectrin. Here, we show that the activation of Plasmodium falciparum SERA6 involves a second, autocatalytic step that is triggered by SUB1 cleavage. Unexpectedly, autoproteolytic maturation of SERA6 requires interaction in multimolecular complexes with a distinct PV‐located protein cofactor, MSA180, that is itself a SUB1 substrate. Genetic ablation of MSA180 mimics SERA6 disruption, producing a fatal block in β‐spectrin cleavage and RBC rupture. Drug‐like inhibitors of SERA6 autoprocessing similarly prevent β‐spectrin cleavage and egress in both P. falciparum and the emerging zoonotic pathogen P. knowlesi. Our results elucidate the egress pathway and identify SERA6 as a target for a new class of antimalarial drugs designed to prevent disease progression.     

Divergent signaling pathways in phagocytic cells during sepsis

Neutrophil accumulation in the lung plays a pivotal role in the pathogenesis of acute lung injury during sepsis. Directed movement of neutrophils is mediated by a group of chemoattractants, especially CXC chemokines. Local lung production of CXC chemokines is intensified during experimental sepsis induced by cecal ligation and puncture (CLP), as reflected by rising levels of MIP-2 and cytokine-induced neutrophil chemoattractant-1 in bronchoalveolar lavage fluids. Alveolar macrophages are primed and blood neutrophils are down-regulated for production of MIP-2 and cytokine-induced neutrophil chemoattractant production in response to LPS and C5a. Under these conditions of stimulation, activation of MAPKs (p38, p42/p44) occurs in sham neutrophils but not in CLP neutrophils, while under the same conditions phosphorylation of p38 and p42/p44 occurs in both sham and CLP alveolar macrophages. These data indicate that, under septic conditions, there is impaired signaling in neutrophils and enhanced signaling in alveolar macrophages, resulting in CXC chemokine production, and C5a appears to play a pivotal role in this process. As a result, CXC chemokines increase in lung, setting the stage for neutrophil accumulation in lung during sepsis.