A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides

Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly alpha-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation.

Molecular basis of Yersinia enterocolitica temperature-dependent resistance to antimicrobial peptides

Antimicrobial peptides (APs) belong to the arsenal of weapons of the innate immune system against infections. In the case of gram-negative bacteria, APs interact with the anionic lipid A moiety of the lipopolysaccharide (LPS). In yersiniae most virulence factors are temperature regulated. Studies from our laboratory demonstrated that Yersinia enterocolitica is more susceptible to polymyxin B, a model AP, when grown at 37°C than at 22°C (J. A. Bengoechea, R. Díaz, and I. Moriyón, Infect. Immun. 64:4891-4899, 1996), and here we have extended this observation to other APs, not structurally related to polymyxin B. Mechanistically, we demonstrate that the lipid A modifications with aminoarabinose and palmitate are downregulated at 37°C and that they contribute to AP resistance together with the LPS O-polysaccharide. Bacterial loads of lipid A mutants in Peyer's patches, liver, and spleen of orogastrically infected mice were lower than those of the wild-type strain at 3 and 7 days postinfection. PhoPQ and PmrAB two-component systems govern the expression of the loci required to modify lipid A with aminoarabinose and palmitate, and their expressions are also temperature regulated. Our findings support the notion that the temperature-dependent regulation of loci controlling lipid A modifications could be explained by H-NS-dependent negative regulation alleviated by RovA. In turn, our data also demonstrate that PhoPQ and PmrAB regulate positively the expression of rovA, the effect of PhoPQ being more important. However, rovA expression reached wild-type levels in the phoPQ pmrAB mutant background, hence indicating the existence of an unknown regulatory network controlling rovA expression in this background.

Molecular dynamics studies of trinucleotide repeat DNA involved in neurodegenerative disorders.

Expansion of trinucleotide repeat DNA of the classes CAG�·CTG, CGG�·CCG and GAA�·TTC are found to be associated with several neurodegenerative disorders. Different mechanisms have been attributed to the expansion of triplets, mainly involving the formation of alternate secondary structures by such repeats. This paper reports the molecular dynamics simulation of triplet repeat DNA sequences to study the basic structural features of DNA that are responsible for the formation of structures such as hairpins and slip-strand DNA leading to expansion. All the triplet repeat sequences studied were found to be more flexible compared to the control sequence unassociated with disease. Moreover, flexibility was found to be in the order CAG�·CTG > CGG�·CCG = GAA�·TTC, the highly flexible CAG�·CTG repeat being the most common cause of neurodegenerative disorders. In another simulation, a single G�·C to T�·A mutation at the 9th position of the CAG�·CTG repeat exhibited a reduction in bending compared to the pure 15-mer CAGâ�¢CTG repeat. EPM1 dodecamer repeat associated with the pathogenesis of progressive myoclonus epilepsy was also simulated and showed flexible nature suggesting a similar expansion mechanism.

3D-QSAR studies on 4-Hydroxyphenylpyruvate Dioxygenase inhibitors by comparative molecular field analysis (CoMFA)

A comparative molecular field analysis (CoMFA) of alkanoic acid 3-oxo-cyclohex-1-enyl ester and 2-acylcyclohexane-1,3-dione derivatives of 4-hydroxyphenylpyruvate dioxygenase inhibitors has been performed to determine the factors required for the activity of these compounds. The substrate's conformation abstracted from dynamic modeling of the enzyme-substrate complex was used to build the initial structures of the inhibitors. Satisfactory results were obtained after an all-space searching procedure, performing a leave-one out (LOO) cross-validation study with cross-validation q(2) and conventional r(2) values of 0.779 and 0.989, respectively. The results provide the tools for predicting the affinity of related compounds, and for guiding the design and synthesis of new HPPD ligands with predetermined affinities.

Molecular mechanisms of measles virus persistence.

As measles virus causes subacute sclerosing panencephalitis and measles inclusion body encephalitis due to its ability to establish human persistent infection, without symptoms for the time between the acute infection and the onset of clinical symptoms, it has been the paradigm for a long term persistent as opposed to chronic infection by an RNA virus. We have reviewed the mechanisms of persistence of the virus and discuss specific mutations associated with CNS infection affecting the matrix and fusion protein genes. These are placed in the context of our current understanding of the viral replication cycle. We also consider the proposed mechanisms of persistence of the virus in replicating cell cultures and conclude that no general mechanistic model can be derived from our current state of knowledge. Finally, we indicate how reverse genetics approaches and the use of mouse models with specific knock-out and knock-in modifications can further our understanding of measles virus persistence.

Impact of antibiotics on conjugational resistance gene transfer in Staphylococcus aureus in sewage.

Ohlsen K, Ternes T, Werner G, Wallner U, Löffler D, Ziebuhr W, Witte W, Hacker J. Institute for Molecular Biology of Infectious Diseases, The University of Würzburg, Röntgenring 11, D-97070 Würzburg, Germany. knut.ohlsen@mail.uni-wuerzburg.de The growing rate of microbial pathogens becoming resistant to standard antibiotics is an important threat to public health. In order to assess the role of antibiotics in the environment on the spread of resistance factors, the impact of subinhibitory concentrations of antibiotics in sewage on gene transfer was investigated using conjugative gentamicin resistance (aacA-aphD) plasmids of Staphylococcus aureus. Furthermore, the concentration of antibiotics in hospital sewage was measured by high-performance liquid chromatography (HPLC)-electrospray tandem mass spectrometry. Several antibiotics were found to be present in sewage, e.g. ciprofloxacin up to 0.051 mgl(-1) and erythromycin up to 0.027 mgl(-1). Resistance plasmid transfer occurred both on solidified (dewatered) sewage and in liquid sewage in a bioreactor with a frequency of 1.1x10(-5)-5.0x10(-8). However, low-level concentrations of antibiotics measured in sewage are below concentrations that can increase plasmid transfer frequencies of gentamicin resistance plasmids of staphylococci.