Anti-Listeria activities of Galleria mellonella hemolymph proteins

We report the use of antimicrobial hemolymph proteins from the model host Galleria mellonella as an inhibitor for various Listeria strains, providing a novel source for antilisterial therapeutics. We also have shown that specific virulence-associated genes known to mediate antimicrobial resistance of Listeria in mammalian models indicated a similar function in Galleria.     

Human Vδ1 γδ T cells expanded from peripheral blood exhibit specific cytotoxicity against B-cell chronic lymphocytic leukemia-derived cells

Background aimsThere is increasing interest in using γδ T cells (GDTC) for cancer immunotherapy. Most studies have been concerned with the Vδ2 subset in blood, for which several expansion protocols exist. We have developed a protocol to expand Vδ1 and Vδ2 preferentially from human blood. We have characterized these subsets and their specificities for leukemic targets.MethodsGDTC were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors via positive magnetic cell sorting; their proliferation in vitro was induced by exposure to the mitogen concanavalin A (Con A). CD107 and cytotoxicity (Cr⁵¹-release and flow cytometric) assays were performed. GDTC clones and target cells were immunophenotyped via flow cytometry.ResultsLonger initial exposure to Con A typically resulted in higher Vδ1 prevalence. Vδ1 were activated by and cytotoxic to B-cell chronic lymphocytic leukemia (B-CLL)-derived MEC1 cells, whereas Vδ2 also responded to MEC1 but more so to the Philadelphia chromosome-positive [Ph+] leukemia cell line EM-enhanced green fluorescent protein (2eGFPluc). Vδ2 clone cytotoxicity against EM-2eGFPluc correlated with Vδ2 T-cell antigen receptor (TCR) and receptor found on Natural Killer cells and many T-cells (NKG2D), whereas Vδ1 clone cytotoxicity versus MEC1 correlated with Vδ1 TCR, CD56 and CD95 expression. Vδ1 also killed Epstein-Barr Virus (EBV)-negative B-CLL-derived TMD2 cells. Immunophenotyping revealed reduced HLA-ABC expression on EM-2eGFPluc, whereas MEC1 and TMD2 exhibited higher Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAILR1).ConclusionsOur ability to expand peripheral Vδ1 cells and show their cytotoxicity to B-CLL-derived cell lines suggests that this novel approach to the cellular treatment of B-CLL may be feasible.     

The MprF protein is required for lysinylation of phospholipids in listerial membranes and confers resistance to cationic antimicrobial peptides (CAMPs) on Listeria monocytogenes

Pathogenic bacteria have to cope with defence mechanisms mediated by adaptive and innate immunity of the host cells. Cationic antimicrobial peptides (CAMPs) represent one of the most effective components of the host innate immune response. Here we establish the function of Lmo1695, a member of the VirR-dependent virulence regulon, recently identified in Listeria monocytogenes. Lmo1695 encodes a membrane protein of 98 kDa with strong homology to the multiple peptide resistance factor (MprF) of Staphylococcus aureus. Like staphylococcal MprF, we found that Lmo1695 is involved in the synthesis of the membrane phospholipid lysylphosphatidylglycerol (L-PG). In addition, Lmo1695 is also essential for lysinylation of diphosphatidylglycerol (DPG), another phospholipid widely distributed in bacterial membranes. A Deltalmo1695 mutant lacking the lysinylated phospholipids was particularly susceptible to CAMPs of human and bacterial origin. The mutant strain infected both epithelial cells and macrophages only poorly and was attenuated for virulence when tested in a mouse model of infection. Lmo1695 is a member of a growing list of survival factors which enable growth of L. monocytogenes in different environments.     

Listeria monocytogenes activated p38 MAPK and induced IL-8 secretion in a nucleotide-binding oligomerization domain 1-dependent manner in endothelial cells

Nucleotide-binding oligomerization domain (Nod) proteins serve as intracellular pattern recognition molecules recognizing peptidoglycans. To further examine intracellular immune recognition, we used Listeria monocytogenes as an organism particularly amenable for studying innate immunity to intracellular pathogens. In contrast to wild-type L. monocytogenes, the nonpathogenic Listeria innocua, or L. monocytogenes mutants lacking internalin B or listeriolysin O, poorly invaded host cells and escaped into host cell cytoplasm, respectively, and were therefore used as controls. In this study, we show that only the invasive wild-type L. monocytogenes, but not the listeriolysin O- or internalin B-negative L. monocytogenes mutants or L. innocua, substantially induced IL-8 production in HUVEC. RNA interference and Nod1-overexpression experiments demonstrated that Nod1 is critically involved in chemokine secretion and NF-kappaB activation initiated by L. monocytogenes in human endothelial cells. Moreover, we show for the first time that Nod1 mediated activation of p38 MAPK signaling induced by L. monocytogenes. Finally, L. monocytogenes- and Nod1-induced IL-8 production was blocked by a specific p38 inhibitor. In conclusion, L. monocytogenes induced a Nod1-dependent activation of p38 MAPK signaling and NF-kappaB which resulted in IL-8 production in endothelial cells. Thus, Nod1 is an important component of a cytoplasmic surveillance pathway.     

Complete sequences of plasmids from the hemolytic-uremic syndrome-associated Escherichia coli strain HUSEC41

The complete and annotated sequences of four plasmids from a historical enteroaggregative Shiga toxin-producing Escherichia coli (HUSEC) serotype O104:H4 strain, HUSEC41/01-09591, isolated in 2001 in Germany are reported.     

The ferritin-like protein Frm is a target for the humoral immune response to Listeria monocytogenes and is required for efficient bacterial survival

The identity and role of listerial antigens recognized by antibodies following listerial infection is largely unknown. We identified the listerial ferritin-like protein Frm as a target of the humoral response following infection of mice with pathogenic Listeria monocytogenes. Specific antibodies to Frm are detected in antisera of mice infected with a pathogenic strain but not in antisera of mice infected with a non-pathogenic L. innocua strain. Antibodies raised to purified listerial ferritin allowed us to demonstrate that the expression of Frm is both growth phase- and temperature-dependent. Using an isogenic Δfrm mutant, we find that ferritin is essential for bacterial growth in chemically defined minimal media but not in complex media such as brain–heart infusion. Mutant bacteria also exhibit a defect in intracellular replication. The Δfrm strain is hypersensitive to hydrogen peroxide indicating that the Frm is required for protection against reactive oxygen intermediates under various growth conditions. Animal studies show that Frm contributes to pathogenesis in mice, in particular, at early time points following infection.     

Rapid Identification and Typing of Listeria Species by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Listeria monocytogenes is a food-borne pathogen that is the causative agent of human listeriosis, an opportunistic infection that primarily infects pregnant women and immunologically compromised individuals. Rapid, accurate discrimination between Listeria strains is essential for appropriate therapeutic management and timely intervention for infection control. A rapid method involving matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) that shows promise for identification of Listeria species and typing and even allows for differentiation at the level of clonal lineages among pathogenic strains of L. monocytogenes is presented. A total of 146 strains of different Listeria species and serotypes as well as clinical isolates were analyzed. The method was compared with the pulsed-field gel electrophoresis analysis of 48 Listeria strains comprising L. monocytogenes strains isolated from food-borne epidemics and sporadic cases, isolates representing different serotypes, and a number of Listeria strains whose genomes have been completely sequenced. Following a short inactivation/extraction procedure, cell material from a bacterial colony was deposited on a sample target, dried, overlaid with a matrix necessary for the MALDI process, and analyzed by MALDI-TOF MS. This technique examines the chemistry of major proteins, yielding profile spectra consisting of a series of peaks, a characteristic “fingerprint” mainly derived from ribosomal proteins. Specimens can be prepared in a few minutes from plate or liquid cultures, and a spectrum can be obtained within 1 minute. Mass spectra derived from Listeria isolates showed characteristic peaks, conserved at both the species and lineage levels. MALDI-TOF MS fingerprinting may have potential for Listeria identification and subtyping and may improve infection control measures.     

The musculotendinous system of an anguilliform swimmer: Muscles, myosepta, dermis, and their interconnections in Anguilla rostrata

Eel locomotion is considered typical of the anguilliform swimming mode of elongate fishes and has received substantial attention from various perspectives such as swimming kinematics, hydrodynamics, muscle physiology, and computational modeling. In contrast to the extensive knowledge of swimming mechanics, there is limited knowledge of the internal body morphology, including the body components that contribute to this function. In this study, we conduct a morphological analysis of the collagenous connective tissue system, i.e., the myosepta and skin, and of the red muscle fibers that sustain steady swimming, focusing on the interconnections between these systems, such as the muscle-tendon and myosepta-skin connections. Our aim is twofold: (1) to identify the morphological features that distinguish this anguilliform swimmer from subcarangiform and carangiform swimmers, and (2) to reveal possible pathways of muscular force transmission by the connective tissue in eels. To detect gradual morphological changes along the trunk we investigated anterior (0.4L), midbody (0.6L), and posterior body positions (0.75L) using microdissections, histology, and three-dimensional reconstructions. We find that eel myosepta have a mediolaterally oriented tendon in each the epaxial and hypaxial regions (epineural or epipleural tendon) and two longitudinally oriented tendons (myorhabdoid and lateral). The latter two are relatively short (4.5-5% of body length) and remain uniform along a rostrocaudal gradient. The skin and its connections were additionally analyzed using scanning electron microscopy (SEM). The stratum compactum of the dermis consists of approximately 30 layers of highly ordered collagen fibers of alternating caudodorsal and caudoventral direction, with fiber angles of 60.51 +/- 7.05 degrees (n = 30) and 57.58 +/- 6.92 degrees (n = 30), respectively. Myosepta insert into the collagenous dermis via fiber bundles that pass through the loose connective tissue of the stratum spongiosum of the dermis and either weave into the layers of the stratum compactum (weaving fiber bundles) or traverse the stratum compactum (transverse fiber bundles). These fiber bundles are evenly distributed along the insertion line of the myoseptum. Red muscles insert into lateral and myorhabdoid myoseptal tendons but not into the horizontal septum or dermis. Thus, red muscle forces might be distributed along these tendons but will only be delivered indirectly into the dermis and horizontal septum. The myosepta-dermis connections, however, appear to be too slack for efficient force transmission and collagenous connections between the myosepta and the horizontal septum are at obtuse angles, a morphology that appears inadequate for efficient force transmission. Though the main modes of undulatory locomotion (anguilliform, subcarangiform, and carangiform) have recently been shown to be very similar with respect to their midline kinematics, we are able to distinguish two morphological classes with respect to the shape and tendon architecture of myosepta. Eels are similar to subcarangiform swimmers (e.g., trout) but are substantially different from carangiform swimmers (e.g., mackerel). This information, in addition to data from kinematic and hydrodynamic studies of swimming, shows that features other than midline kinematics (e.g., wake patterns, muscle activation patterns, and morphology) might be better for describing the different swimming modes of fishes.     

Lipoproteins of Listeria monocytogenes are critical for virulence and TLR2-mediated immune activation

Numerous cell surface components of Listeria influence and regulate innate immune recognition and virulence. Here, we demonstrate that lipidation of prelipoproteins in Listeria monocytogenes is required to promote NF-kappaB activation via TLR2. In HeLa cells transiently expressing TLR2, L. monocytogenes and Listeria innocua mutants lacking the prolipoprotein diacylglyceryl transferase (lgt) gene are unable to induce TLR2-dependent activation of NF-kappaB, a property intrinsic to their isogenic parental strains. TLR2-dependent immune recognition is directed to secreted, soluble lipoproteins as evidenced by the sensitivity of the response to lipoprotein lipase. Studies of bone marrow-derived macrophages of C57BL/6 wild-type and TLR2-deficient mice infected with wild-type and lgt mutant strains indicate that the absence of host TLR2 receptor signaling has consequences similar to those of the absence of the bacterial TLR2 ligand, i.e., a delay in cellular immune responses directed toward the bacterium. Infection studies with the wild-type and TLR2(-/-) mice indicated attenuation of the lgt deletion mutant in both mouse strains, implying multiple roles of lipoproteins during infection. Further characterization of the Delta lgt mutant indicated that it is impaired for both invasion and intracellular survival and exhibits increased susceptibility to cationic peptides. Our studies identify lipoproteins as the immunologically active ligand of TLR2 and assign a critical role for this receptor in the recognition of these bacteria during infection, but they also reveal the overall importance of the lipoproteins for the pathogenicity of Listeria.     

Psychological assessment in XLMR: a proposal for setting international standards

The need for an agreed upon set of standards for assessing individuals with XLMR was made quite evident this past year at the Fragile X and XLMR Workshop in Strasbourg. Several affected individuals from different families may have been incorrectly diagnosed as MR. Many factors can have a negative affect on IQ testing. As a result, evaluating individuals with cognitive deficits can be problematic. To be effective, psychological assessments must produce uniform results that are consistent with the definition of MR. Therefore, to foster international research in XLMR. I propose a two-stage standardized protocol. To determine which tests may be suitable. I review an assortment of instruments for psychological assessment at each stage, noting their strengths and weaknesses. Afterward, I present a set of standardized protocols based on age and language ability.