Paenibacillus larvae Chitin-Degrading Protein PlCBP49 Is a Key Virulence Factor in American Foulbrood of Honey Bees

Paenibacillus larvae, the etiological agent of the globally occurring epizootic American Foulbrood (AFB) of honey bees, causes intestinal infections in honey bee larvae which develop into systemic infections inevitably leading to larval death. Massive brood mortality might eventually lead to collapse of the entire colony. Molecular mechanisms of host-microbe interactions in this system and of differences in virulence between P. larvae genotypes are poorly understood. Recently, it was demonstrated that the degradation of the peritrophic matrix lining the midgut epithelium is a key step in pathogenesis of P. larvae infections. Here, we present the isolation and identification of PlCBP49, a modular, chitin-degrading protein of P. larvae and demonstrate that this enzyme is crucial for the degradation of the larval peritrophic matrix during infection. PlCBP49 contains a module belonging to the auxiliary activity 10 (AA10, formerly CBM33) family of lytic polysaccharide monooxygenases (LPMOs) which are able to degrade recalcitrant polysaccharides. Using chitin-affinity purified PlCBP49, we provide evidence that PlCBP49 degrades chitin via a metal ion-dependent, oxidative mechanism, as already described for members of the AA10 family. Using P. larvae mutants lacking PlCBP49 expression, we analyzed in vivo biological functions of PlCBP49. In the absence of PlCBP49 expression, peritrophic matrix degradation was markedly reduced and P. larvae virulence was nearly abolished. This indicated that PlCBP49 is a key virulence factor for the species P. larvae. The identification of the functional role of PlCBP49 in AFB pathogenesis broadens our understanding of this important family of chitin-binding and -degrading proteins, especially in those bacteria that can also act as entomopathogens.     

Molecular identification, immunolocalization, and functional activity of a vacuolar-type H+-ATPase in bovine rumen epithelium

In this study, we have studied the expression, localization, and functionality of vacuolar-type H⁺-ATPase (vH⁺-ATPase) and Na⁺/K⁺-ATPase in the bovine rumen epithelium. Compared with the intracellular pH (pH_i) of control rumen epithelial cells (REC; 7.06 ± 0.07), application of inhibitors selective for vH⁺-ATPase (foliomycin) and Na⁺/K⁺-ATPase (ouabain) reduced pH_i by 0.10 ± 0.03 and 0.18 ± 0.03 pH-units, respectively, thereby verifying the existence of both functional proteins. Results from qRT-PCR and immunoblotting clearly confirm the expression of vH⁺-ATPase B subunit in REC. However, the amount of Na⁺/K⁺-ATPase mRNA and protein is tenfold and 11-fold of those of vH⁺-ATPase subunit B, respectively, reflecting a lower overall abundance of the latter in REC. Na⁺/K⁺-ATPase immunostaining has revealed the protein in the plasma membrane of all REC from the stratum basale to stratum granulosum, with the highest abundance in basal cells. In contrast, the vH⁺-ATPase B subunit has been detected in groups of cells only, mainly localized in the stratum spinosum and stratum granulosum of the epithelium. Furthermore, vH⁺-ATPase has been detected in the cell membrane and in intracellular pools. Thus, functional vacuolar-type H⁺ pumps are expressed in REC and probably play a role in the adaptation of epithelial transport processes.     

Growth Monitoring: A Survey of Current Practices of Primary Care Paediatricians in Europe

Objective

We aimed to study current practices in growth monitoring by European primary care paediatricians and to explore their perceived needs in this field.

Methods

We developed a cross-sectional, anonymous on-line survey and contacted primary care paediatricians listed in national directories in the 18 European countries with a confederation of primary care paediatricians. Paediatricians participated in the survey between April and September 2011.

Results

Of the 1,198 paediatricians from 11 European countries (response rate 13%) who participated, 29% used the 2006 World Health Organization Multicentre Growth Reference Study growth charts, 69% used national growth charts; 61% used software to draw growth charts and 79% did not use a formal algorithm to detect abnormal growth on growth charts. Among the 21% of paediatricians who used algorithms, many used non-algorithmic simple thresholds for height and weight and none used the algorithms published in the international literature. In all, 69% of paediatricians declared that a validated algorithm to monitor growth would be useful in daily practice. We found important between-country variations.

Conclusion

The varied growth-monitoring practices declared by primary care paediatricians reveals the need for standardization and evidence-based algorithms to define abnormal growth and the development of software that would use such algorithms.     

Impact of pre-existing MSP142-allele specific immunity on potency of an erythrocytic Plasmodium falciparum vaccine

ABSTRACT: BACKGROUND: MSP1 is the major surface protein on merozoites and a prime candidate for a blood stage malaria vaccine. Preclinical and seroepidemiological studies have implicated antibodies to MSP1 in protection against blood stage parasitaemia and/or reduced parasite densities, respectively. Malaria endemic areas have multiple strains of Plasmodium falciparum circulating at any given time, giving rise to complex immune responses, an issue which is generally not addressed in clinical trials conducted in non-endemic areas. A lack of understanding of the effect of pre-existing immunity to heterologous parasite strains may significantly contribute to vaccine failure in the field. The purpose of this study was to model the effect of pre-existing immunity to MSP142 on the immunogenicity of blood-stage malaria vaccines based on alternative MSP1 alleles. METHODS: Inbred and outbred mice were immunized with various recombinant P. falciparum MSP142 proteins that represent the two major alleles of MSP142, MAD20 (3D7) and Wellcome (K1, FVO). Humoral immune responses were analysed by ELISA and LuminexTM, and functional activity of induced MSP142-specific antibodies was assessed by growth inhibition assays. Tcell responses were characterized using ex vivo ELISpot assays. RESULTS: Analysis of the immune responses induced by various immunization regimens demonstrated a strong allele-specific response at the T cell level in both inbred and outbred mice. The success of heterologous regimens depended on the degree of homology of the N-terminal p33 portion of the MSP142, likely due to the fact that most T cell epitopes reside in this part of the molecule. Analysis of humoral immune responses revealed a marked cross-reactivity between the alleles. Functional analyses showed that some of the heterologous regimens induced antibodies with improved growth inhibitory activities. CONCLUSION: The development of a more broadly efficacious MSP1 based vaccine may be hindered by clonally imprinted p33 responses mainly restricted at the T cell level. In this study, the homology of the p33 sequence between the clonally imprinted response and the vaccine allele determines the magnitude of vaccine induced responses.     

Biotransformation and reduction of estrogenicity of bisphenol A by the biphenyl-degrading Cupriavidus basilensis

The biphenyl-degrading Gram-negative bacterium Cupriavidus basilensis (formerly Ralstonia sp.) SBUG 290 uses various aromatic compounds as carbon and energy sources and has a high capacity to transform bisphenol A (BPA), which is a hormonally active substance structurally related to biphenyl. Biphenyl-grown cells initially hydroxylated BPA and converted it to four additional products by using three different transformation pathways: (a) formation of multiple hydroxylated BPA, (b) ring fission, and (c) transamination followed by acetylation or dimerization. Products of the ring fission pathway were non-toxic and all five products exhibited a significantly reduced estrogenic activity compared to BPA. Cell cultivation with phenol and especially in nutrient broth (NB) resulted in a reduced biotransformation rate and lower product quantities, and NB-grown cells did not produce all five products in detectable amounts. Thus, the question arose whether enzymes of the biphenyl degradation pathway are involved in the transformation of BPA and was addressed by proteomic analyses.

     

Müller glial cell-provided cellular light guidance through the vital guinea-pig retina

In vertebrate eyes, images are projected onto an inverted retina where light passes all retinal layers on its way to the photoreceptor cells. Light scattering within this tissue should impair vision. We show that radial glial (Müller) cells in the living retina minimize intraretinal light scatter and conserve the diameter of a beam that hits a single Müller cell endfoot. Thus, light arrives at individual photoreceptors with high intensity. This leads to an optimized signal/noise ratio, which increases visual sensitivity and contrast. Moreover, we show that the ratio between Müller cells and cones-responsible for acute vision-is roughly 1. This suggests that high spatiotemporal resolution may be achieved by each cone receiving its part of the image via its individual Müller cell-light guide.     

Oval cell proliferation in p16INK4a expressing mouse liver is triggered by chronic growth stimuli.

Terminal differentiation requires molecules also involved in aging such as the cell cycle inhibitor p16(INK4a).Like other organs, the adult liver represents a quiescent organ with terminal differentiated cells, hepatocytes and cholangiocytes. These cells retain the ability to proliferate in response to liver injury or reduction of liver mass. However, under conditions which prevent mitotic activation of hepatocytes, regeneration can occur instead from facultative hepatic stem cells.For therapeutic application a non-toxic activation of this stem cell compartment is required. We have established transgenic mice with conditional overexpression of the cell cycle inhibitor p16(INK4a) in hepatocytes and have provoked and examined oval cell activation in adult liver in response to a range of proliferative stimuli.We could show that the liver specific expression of p16(INK4a) leads to a faster differentiation of hepatocytes and an activation of oval cells already in postnatal mice without negative consequences on liver function.     

From dormant to germinating spores of Streptomyces coelicolor A3(2): new perspectives from the crp null mutant

The complete understanding of the morphological differentiation of streptomycetes is an ambitious challenge as diverse sensors and pathways sensitive to various environmental stimuli control the process. Germination occupies a particular position in the life cycle as the good achievement of the process depends on events occurring both during the preceding sporulation and during germination per se. The cyclic AMP receptor protein (crp) null mutant of Streptomyces coelicolor, affected in both sporulation and germination, was therefore presented as a privileged candidate to highlight new proteins involved in the shift from dormant to germinating spores. Our multidisciplinary approach-combining in vivo data, the analysis of spores morphological properties, and a proteome study-has shown that Crp is a central regulatory protein of the life cycle in S. coelicolor; and has identified spores proteins with statistically significant increased or decreased expression that should be listed as priority targets for further investigations on proteins that trigger both ends of the life cycle.     

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.