Surface displaced alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein

Background: Lactic acid bacteria of the genus Lactobacillus and Bifidobacterium are one of the most important health promoting groups of the human intestinal microbiota. Their protective role within the gut consists in out competing invading pathogens for ecological niches and metabolic substrates. Among the features necessary to provide health benefits, commensal microorganisms must have the ability to adhere to human intestinal cells and consequently to colonize the gut. Studies on mechanisms mediating adhesion of lactobacilli to human intestinal cells showed that factors involved in the interaction vary mostly among different species and strains, mainly regarding interaction between bacterial adhesins and extracellular matrix or mucus proteins. We have investigated the adhesive properties of Lactobacillus plantarum, a member of the human microbiota of healthy individuals. Results: We show the identification of a Lactobacillus plantarum LM3 cell surface protein (48 kDa), which specifically binds to human fibronectin (Fn), an extracellular matrix protein. By means of mass spectrometric analysis this protein was identified as the product of the L. plantarum enoA1 gene, coding the EnoA1 alfa-enolase. Surface localization of EnoA1 was proved by immune electron microscopy. In the mutant strain LM3-CC1, carrying the enoA1 null mutation, the 48 kDa adhesin was not anymore detectable neither by anti-enolase Western blot nor by Fn-overlay immunoblotting assay. Moreover, by an adhesion assay we show that LM3-CC1 cells bind to fibronectin-coated surfaces less efficiently than wild type cells, thus demonstrating the significance of the surface displaced EnoA1 protein for the L. plantarum LM3 adhesion to fibronectin. Conclusion: Adhesion to host tissues represents a crucial early step in the colonization process of either pathogens or commensal bacteria. We demonstrated the involvement of the L. plantarum Eno A1 alfa-enolase in Fn-binding, by studying LM3 and LM3-CC1 surface proteins. Isolation of LM3-CC1 strain was possible for the presence of expressed enoA2 gene in the L. plantarum genome, giving the possibility, for the first time to our knowledge, to quantitatively compare adhesion of wild type and mutant strain, and to assess doubtless the role of L. plantarum Eno A1 as a fibronectin binding protein. © 2009 Castaldo et al; licensee BioMed Central Ltd

Analisi dei tassi di crescita pediatrici nel Petauro dello zucchero (Petaurus breviceps) in cattività

Il Petauro dello zucchero (Petaurus breviceps) è un marsupiale australiano entrato da qualche decennio a far parte degli animali da compagnia non convenzionali; nonostante ciò molti sono i campi ancora inesplorati nello studio della sua fisiologia, fra cui molti aspetti nell'ambito pediatrico. Questo studio è stato svolto presso uno degli allevamenti più grandi d'Italia e si prefigge l'obiettivo di rilevare i tassi medi di crescita dei cuccioli di petauro attraverso il confronto fra diverse misurazioni corporee, considerando il periodo intercorrente tra l’uscita dal marsupio (OOP) fino alla ventesima settimana di vita. Lo studio è il primo ad essere effettuato in petauri in cattività da diverse generazioni, e perciò a non essere limitato ad una sola subspecie o condizionato dallo stress che può facilmente presentarsi in soggetti di cattura, influenzando la crescita del cucciolo; è inoltre il primo effettuato senza alcun uso di anestetici. Lo studio si pone l'obiettivo di fornire ulteriori dati su un aspetto importante della pediatria di questa specie, e di dare al clinico un utile supporto pratico di monitoraggio della crescita del paziente cucciolo che si presenta in visita

Inactivation of two genes located in the GPL biosynthetic locus leads to morphological changes in Mycobacterium smegmatis

Glycopeptidolipids (GPLs) are surface exposed molecules found either in saprophytic or in clinically-relevant non-tuberculous mycobacteria. These molecules, which may represent more than 70% of total surface lipids, are required for cell aggregation, sliding motility and biofilm formation and seem to act as surface antigens stimulating host macrophages response. The GPLs structure is conserved among mycobacteria and is made by a common glycosylated lpopeptide core that is variably modified by O-methylation and O-acetylation. In the fast growing Mycobacterium smegmatis, all the genes necessary for GPL biosynthesis are clustered in a single region of 65 kb and most of them have been identified experimentally or by in silico prediction. Here we report the isolation of two mutant strains of M. smegmatis, carrying null mutations in genes coding for two hypothetical proteins. The two genes (MSMEG_0412; MSMEG_0394) belong to the GPL gene cluster. Preliminary experiments show that inactivation of both genes has drastic impact on bacterial surface properties. Both mutants display rough phenotype, lack of sliding motility, altered biofilm formation and increased antimicrobial susceptibility. GPLs analysis by TLC and MALDI-TOF is in progress to assess whether the observed phenotypes arise from GPLs deficiency and/or structural modification

Transcriptional analysis of exopolysaccharides biosynthesis gene clusters in Lactobacillus plantarum

Exopolysaccharides (EPS) from lactic acid bacteria contribute to specific rheology and texture of fermented milk products and find applications also in non-dairy foods and in therapeutics. Recently, four clusters of genes (cps) associated with surface polysaccharide production have been identified in Lactobacillus plantarum WCFS1, a probiotic and food-associated lactobacillus. These clusters are involved in cell surface architecture and probably in release and/or exposure of immunomodulating bacterial molecules. Here we show a transcriptional analysis of these clusters. Indeed, RT-PCR experiments revealed that the cps loci are organized in five operons. Moreover, by reverse transcription–qPCR analysis performed on L. plantarum WCFS1 (wild type) and WCFS1-2 (ΔccpA), we demonstrated that expression of three cps clusters is under the control of the global regulator CcpA. These results, together with the identification of putative CcpA target sequences (catabolite responsive element CRE) in the regulatory region of four out of five transcriptional units, strongly suggest for the first time a role of the master regulator CcpA in EPS gene transcription among lactobacilli

Effect of inactivation of ccpA and aerobic growth in Lactobacillus plantarum: A proteomic perspective

Lactobacillus plantarum is a facultative heterofermentative lactic acid bacterium widely used in the production of most fermented food due to its ability to thrive in several environmental niches, including the human gut. In order to cope with different growth conditions, it has developed complex molecular response mechanisms, characterized by the induction of a large set of proteins mainly regulated by HrcA and CtsR repressors as well as by global regulators such as carbon catabolite control protein A (CcpA). In this study, the role of CcpA in the regulation of growth under anaerobiosis and aerobiosis, and the adaptation to aeration in L. plantarum WCFS1 were comprehensively investigated by differential proteomics. The inactivation of ccpA, in both growth conditions, significantly changed the expression level of 76 proteins, mainly associated with carbohydrate and energy metabolism, membrane transport, nucleotide metabolism, protein biosynthesis and folding. The role of CcpA as pleiotropic regulator was particularly evident at the shift from homolactic fermentation to mixed fermentation. Proteomic results also indicated that the mutant strain was more responsive to aerobic growth condition

Comparisons among RHIBE schemes.

<p>Comparisons among RHIBE schemes.</p