Inhibitory activities of three classes of acyclic nucleoside phosphonates against murine polyomavirus and primate simian virus 40 strains.

Murine polyomavirus and simian virus 40 were used to evaluate the potencies of the compounds of three classes of acyclic nucleoside phosphonates: (i) the original HPMP (3-hydroxy-2-phosphonomethoxypropyl) and PME (2-phosphonomethoxyethyl) derivatives, (ii) the 6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidine (DAPy) derivatives, and (iii) a new class of HPMP derivatives containing a 5-azacytosine moiety. The last class showed the highest activities and selectivities against both polyomaviruses.

In vitro evaluation of the anti-orf virus activity of alkoxyalkyl esters of CDV, cCDV and (S)-HPMPA.

Acyclic nucleoside phosphonates (ANPs) and in particular (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC, cidofovir, CDV, Vistide) and its adenine counterpart (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine [(S)-HPMPA] are highly active against orf virus infections. This parapoxvirus commonly causes infection in sheep, goats, but also humans. Alkoxyalkyl esters of CDV have an increased oral bioavailability and are more active against orthopoxviruses than the parent compounds. In the present study, the potency of several alkoxyalkyl esters of CDV, cyclic cidofovir (cCDV) and (S)-HPMPA was evaluated against different orf virus isolates in two cell types, human embryonic lung (HEL) fibroblast and primary lamb keratinocytes. Each prodrug was at least 10-fold more active than its parent compound in both cell types. Of all the compounds tested, the (S)-HPMPA alkoxyalkyl esters showed the highest activity and selectivity against orf virus. Our results support the development of alkoxyalkyl esters of ANPs as antivirals not only for the treatment of complicated human orf lesions, but also in the therapy and prophylaxis of contagious ecthyma in sheep and goats.

Detection and quantification of Legionella pneumophila in water samples using competitive PCR.

The presence of high levels of Legionella pneumophila in man-made aquatic systems correlates with the incidence of nosocomial Legionnaires' disease. This requires a rapid, reliable, and sensitive quantification of L. pneumophila concentrations in suspected water systems. In this research, a homologous competitor was developed and evaluated in a L. pneumophila competitive polymerase chain reaction (cPCR) to quantify this human pathogen in a quick, cost-effective, and reliable way. Accuracy of cPCR was evaluated by analyzing cooling tower and tap water samples spiked with known concentrations of L. pneumophila bacteria, in parallel with the standard culture method. Legionella pneumophila amounts detected and calculated from cPCR and culture correlated very well: r = 0.998, P = 0.002 for tap water and r = 0.990, P = 0.009 for cooling tower water. Nevertheless, for both kinds of water samples, mean numbers of L. pneumophila calculated from cPCR results were always higher than those obtained by culture. This study makes it clear that the rapid, sensitive, and cost-effective L. pneumophila cPCR is a promising alternative to the standard time-consuming culture method and expensive real-time PCR to enumerate L. pneumophila bacteria in environmental water samples.

Activities of alkoxyalkyl esters of cidofovir (CDV), cyclic CDV, and (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine against orthopoxviruses in cell monolayers and in organotypic cultures.

The potencies of several alkoxyalkyl esters of acyclic nucleoside phosphonates against vaccinia virus and cowpox virus were evaluated in cell monolayers and three-dimensional epithelial raft cultures. Prodrugs were at least 20-fold more active than their parent compounds. Octadecycloxyethyl-(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine emerged as the most potent derivative.

First proteomic analysis of Legionella pneumophila based on its developing genome sequence.

The first proteomic analysis of the respiratory pathogen Legionella pneumophila ATCC 33152 is presented in this report. Two-dimensional gel electrophoresis of total cell extracts was carried out. In total, 130 protein spots were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MS) or by quadruple time-of-flight tandem MS, including proteins correlated with virulence. For the first time, proteins of L. pneumophila were identified using mass spectrometric methods and mapped on a two-dimensional gel; this will be of considerable use for comparison of protein expression profiles of L. pneumophila wild-type and knock-out mutant strains and of L. pneumophila grown under different conditions.

The use of the cMyc epitope tag can be problematic for protein detection in Legionella pneumophila.

In various recent experiments with respect to protein secretion by L. pneumophila, protein detection based on the cMyc-tag seemed to be problematic. For one specific protein, we studied the reason for this problem in more detail by comparing protein expression in the presence and absence of the cMyc-tag. We demonstrated a decrease in the amount of mRNA transcript when the cMyc coding sequence is present. We can conclude that this epitope tag is not a preferential tag for protein detection in L. pneumophila.

Molecular and functional characterization of type I signal peptidase from Legionella pneumophila.

Legionella pneumophila is a facultative intracellular Gram-negative rod-shaped bacterium that has become an important cause of both community-acquired and nosocomial pneumonia. Numerous studies concerning the unravelling of the virulence mechanism of this important pathogen have been initiated. As evidence is now accumulating for the involvement of protein secretion systems in bacterial virulence in general, the type I signal peptidase (LepB) of L. pneumophila was of particular interest. This endopeptidase plays an essential role in the processing of preproteins carrying a typical amino-terminal signal peptide, upon translocation across the cytoplasmic membrane. This paper reports the cloning and the transcriptional analysis of the L. pneumophila lepB gene encoding the type I signal peptidase (SPase). Reverse transcription PCR experiments showed clear lepB expression when L. pneumophila was grown both in culture medium, and also intracellularly in Acanthamoeba castellanii, a natural eukaryotic host of L. pneumophila. In addition, LepB was shown to be encoded by a polycistronic mRNA transcript together with two other proteins, i.e. a LepA homologue and a ribonuclease III homologue. SPase activity of the LepB protein was demonstrated by in vivo complementation analysis in a temperature-sensitive Escherichia coli lepB mutant. Protein sequence and predicted membrane topology were compared to those of leader peptidases of other Gram-negative human pathogens. Most strikingly, a strictly conserved methionine residue in the substrate binding pocket was replaced by a leucine residue, which might influence substrate recognition. Finally it was shown by in vivo experiments that L. pneumophila LepB is a target for (5S,6S)-6-[(R)-acetoxyethyl]-penem-3-carboxylate, a specific inhibitor of type I SPases.

A putative twin-arginine translocation pathway in Legionella pneumophila.

Legionella pneumophila is a facultative intracellular human pathogen causing Legionnaires' disease, a severe form of pneumonia. Because of the importance of secretion pathways in virulence, we were interested in the possible presence of the twin-arginine translocation (Tat) pathway in L. pneumophila. This secretion pathway is used to transport folded proteins, characterized by two arginines in their signal peptide, across the cytoplasmic membrane. We describe here the presence of a putative Tat pathway in L. pneumophila. Three genes encoding Escherichia coli TatA, TatB, and TatC homologues were identified. The tatA and tatB genes were shown to constitute an operon while tatC is monocistronic. RT-PCR analysis revealed expression of the tat genes during both exponential and stationary growth as well as during intracellular growth in Acanthamoeba castellanii. A search for the conserved twin-arginine motif in predicted signal peptides resulted in a list of putative Tat substrates.

Novel transcriptional regulators of Legionella pneumophila that affect replication in Acanthamoeba castellanii.

Legionella pneumophila is commonly found in freshwater environments and is able to invade and replicate within amoebae and ciliated protozoa. Moreover, this bacterium is also able to replicate within human alveolar macrophages causing a severe form of pneumonia, designated Legionnaires' disease. L. pneumophila pathogenesis is not yet completely understood, but the genes responsible for infection and intracellular replication are becoming known. Nonetheless, knowledge as to how these genes are controlled is still very limited. The partially sequenced genome of L. pneumophila was searched for open reading frames encoding proteins with sequence similarity to members of the LuxR family of transcriptional regulators. These were designated LpnR1, LpnR2, LpnR3, and LpnR4. Although these proteins could not be identified as true LuxR proteins, they do act as regulators, as illustrated in this report. LpnR1 negatively affected rpoS expression, whereas LpnR2 and LpnR3 positively affected flagellin expression. Furthermore, LpnR2 proved to be necessary for efficient invasion of Acanthamoeba castellanii and LpnR3 for intracellular replication in this protozoan host. LpnR4 was recently identified as LetA.

Neurotoxic and neurobehavioral effects of kynurenines in adult insects.

Kynurenines are endogenous metabolites of tryptophan, which are studied extensively in vertebrates with respect to their etiological role in the pathology of various neurodegenerative disorders. In insects, metabolites of the kynurenic pathway are present in peak concentrations in the hemolymph of holometabolic species during pupation and just before eclosion. Unlike in larvae, these compounds cause severe motor dysfunction in adult species. Adult flesh flies were injected with various concentrations of these endogenous toxins and the effects on motor function were assessed. For tryptophan, L-kynurenine, 3-hydroxy-kynurenine, and anthranilic acid, the effects ranged from reversible to irreversible motor dysfunction, to instant paralysis and death. 3-Hydroxy-anthranilic acid could induce a tetanus like spasm of the wings. Tryptophan, 3-hydroxykynurenine, and 3-hydroxy-anthranilic acid were toxic to primary cultures of insect neurons. It is possible that some of these metabolites have a distinct role in larvae during the apoptotic events related to neurometamorphosis.