Novel Immunoprotective Proteins of Streptococcus pneumoniae Identified by Opsonophagocytosis Killing Screen.

The success of polysaccharide conjugate vaccines represents a major advance in the prevention of pneumococcal disease, but the power of these vaccines is limited by partial spectrum of coverage and high cost. Vaccines using immunoprotective proteins are a promising alternative type of pneumococcal vaccines. In this study, we constructed a library of antisera against conserved pneumococcal proteins predicted to be associated with cell surface or virulence using a combination of bioinformatic prediction and immunization of rabbits with recombinant proteins. Screening of the library by an opsonophagocytosis killing (OPK) assay identified the OPK-positive antisera, which represented 15 (OPK-positive) proteins. Further tests showed that virtually all of these OPK-positive antisera conferred passive protection against lethal infection of virulent pneumococci. More importantly, immunization with recombinant forms of three OPK-positive proteins (SP148, PBP2b, and ScpB), alone or in combination, conferred significant protection against lethal challenge of pneumococcal strains representing capsular serotypes 3, 4, and 6A in a mouse sepsis model. To our best knowledge, this work represents the first example in which novel vaccine candidates are successfully identified by the OPK screening. Our data have also provided further confirmation that the OPK activity may serve as a reliable in vitro surrogate for evaluating vaccine efficacy of pneumococcal proteins.

Identification of the lipid biomarkers from plasma in idiopathic pulmonary fibrosis by Lipidomics.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is an irreversible interstitial pulmonary disease featured by high mortality, chronic and progressive course, and poor prognosis with unclear etiology. Currently, more studies have been focusing on identifying biomarkers to predict the progression of IPF, such as genes, proteins, and lipids. Lipids comprise diverse classes of molecules and play a critical role in cellular energy storage, structure, and signaling. The role of lipids in respiratory diseases, including cystic fibrosis, asthma and chronic obstructive pulmonary disease (COPD) has been investigated intensely in the recent years. The human serum lipid profiles in IPF patients however, have not been thoroughly understood and it will be very helpful if there are available molecular biomarkers, which can be used to monitor the disease progression or provide prognostic information for IPF disease. METHODS: In this study, we performed the ultraperformance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-QTOF/MS) to detect the lipid variation and identify biomarker in plasma of IPF patients. The plasma were from 22 IPF patients before received treatment and 18 controls. RESULTS: A total of 507 individual blood lipid species were determined with lipidomics from the 40 plasma samples including 20 types of fatty acid, 159 types of glycerolipids, 221 types of glycerophospholipids, 47 types of sphingolipids, 46 types of sterol lipids, 7 types of prenol lipids, 3 types of saccharolipids, and 4 types of polyketides. By comparing the variations in the lipid metabolite levels in IPF patients, a total of 62 unique lipids were identified by statistical analysis including 24 kinds of glycerophoslipids, 30 kinds of glycerolipids, 3 kinds of sterol lipids, 4 kinds of sphingolipids and 1 kind of fatty acids. Finally, 6 out of 62 discriminating lipids were selected as the potential biomarkers, which are able to differentiate between IPF disease and controls with ROC analysis. CONCLUSIONS: Our results provided vital information regarding lipid metabolism in IPF patients and more importantly, a few potentially promising biomarkers were firstly identified which may have a predictive role in monitoring and diagnosing IPF disease.

Allelic Variation of the Capsule Promoter Diversifies Encapsulation and Virulence In Streptococcus pneumoniae.

The polysaccharide capsule is the major virulence factor of Streptococcus pneumoniae (pneumococcus), a major human pathogen. The sequences in the promoter and coding regions of the capsule gene locus undergo extensive variations through the natural transformation-mediated horizontal gene transfer. The sequence variations in the coding region have led to at least 97 capsular serotypes. However, it remains unclear whether the sequence polymorphisms in the promoter region have any biological significance. In this study, we determined the sequences of the cps promoter region from 225 invasive pneumococcal isolates, and identified modular composition and remarkable inter-strain sequence variations in this region. The strain-to strain variations in the cps promoter are characterized by diversity in sequence and size, mosaic combinations of nucleotide polymorphisms and sequence modules, selective preservation of the sequence combinations, and promiscuous assortments of the sequences between the promoter and coding regions. Isogenic pneumococci carrying allelic variants of the cps promoter displayed significant differences in the transcription of the capsule genes, capsule production, adhesion to host epithelial cells, anti-phagocytosis and virulence in mouse bacteremia model. This study has thus indicated that the sequence polymorphisms in the cps promoter represent a novel mechanism for fine-tuning the level of encapsulation and virulence among S. pneumoniae strains.

Sequence elements upstream of the core promoter are necessary for full transcription of the capsule gene operon in Streptococcus pneumoniae strain D39.

Streptococcus pneumoniae is a major bacterial pathogen in humans. Its polysaccharide capsule is a key virulence factor that promotes bacterial evasion of human phagocytic killing. While S. pneumoniae produces at least 94 antigenically different types of capsule, the genes for biosynthesis of almost all capsular types are arranged in the same locus. The transcription of the capsular polysaccharide (cps) locus is not well understood. This study determined the transcriptional features of the cps locus in the type 2 virulent strain D39. The initial analysis revealed that the cps genes are cotranscribed from a major transcription start site at the -25 nucleotide (G) upstream of cps2A, the first gene in the locus. Using unmarked chromosomal truncations and a luciferase-based transcriptional reporter, we showed that the full transcription of the cps genes not only depends on the core promoter immediately upstream of cps2A, but also requires additional elements upstream of the core promoter, particularly a 59-bp sequence immediately upstream of the core promoter. Unmarked deletions of these promoter elements in the D39 genome also led to significant reduction in CPS production and virulence in mice. Lastly, common cps gene (cps2ABCD) mutants did not show significant abnormality in cps transcription, although they produced significantly less CPS, indicating that the CpsABCD proteins are involved in the encapsulation of S. pneumoniae in a posttranscriptional manner. This study has yielded important information on the transcriptional characteristics of the cps locus in S. pneumoniae.

Structure of pneumococcal peptidoglycan hydrolase LytB reveals insights into the bacterial cell wall remodeling and pathogenesis.

Streptococcus pneumoniae causes a series of devastating infections in humans. Previous studies have shown that the endo-ß-N-acetylglucosaminidase LytB is critical for pneumococcal cell division and nasal colonization, but the biochemical mechanism of LytB action remains unknown. Here we report the 1.65 Å crystal structure of the catalytic domain (residues Lys-375-Asp-658) of LytB (termed LytBCAT), excluding the choline binding domain. LytBCAT consists of three structurally independent modules: SH3b, WW, and GH73. These modules form a "T-shaped" pocket that accommodates a putative tetrasaccharide-pentapeptide substrate of peptidoglycan. Structural comparison and simulation revealed that the GH73 module of LytB harbors the active site, including the catalytic residue Glu-564. In vitro assays of hydrolytic activity indicated that LytB prefers the peptidoglycan from the lytB-deficient pneumococci, suggesting the existence of a specific substrate of LytB in the immature peptidoglycan. Combined with in vitro cell-dispersing and in vivo cell separation assays, we demonstrated that all three modules are necessary for the optimal activity of LytB. Further functional analysis showed that the full catalytic activity of LytB is required for pneumococcal adhesion to and invasion into human lung epithelial cells. Structure-based alignment indicated that the unique modular organization of LytB is highly conserved in its orthologs from Streptococcus mitis group and Gemella species. These findings provided structural insights into the pneumococcal cell wall remodeling and novel hints for the rational design of therapeutic agents against pneumococcal growth and thereby the related diseases.

Indian mustard aquaporin improves drought and heavy-metal resistance in tobacco.

An aquaporin cDNA BjPIP1 isolated from heavy-metal accumulator Indian mustard (Brassica juncea L.) encodes a 286-residue protein. The deduced amino acid sequence of BjPIP1 with six putative transmembrane domains showed highest identity (85-99%) to PIP1 subfamily members. Semi-quantitative RT-PCR analysis revealed that BjPIP1 transcripts were more abundantly expressed in roots compared to aerial parts of Indian mustard. However, the expression of BjPIP1 in leaves was up-regulated by drought, salt, low temperature, and heavy metal stress, suggesting that BjPIP1 was involved in resistance to abiotic stresses. BjPIP1 under the control of 35S promoter was introduced into tobacco mediated with Agrobacterium tumefaciens, the transgenic tobacco exhibited a lower water loss rate, a decreased transpiration rate, and stomatal conductance compared to the wild-type plants under osmotic stress, indicating that BjPIP1 might enhance plant drought resistance by decreasing transpiration via reducing stomatal conductance. Furthermore, overexpression of BjPIP1 in tobacco enhanced Cd resistance of root growth, and lowered transpiration rate and stomatal conductance upon Cd exposure, suggesting that BjPIP1 might increase heavy-metal resistance by maintaining reasonable water status in tobacco. Moreover, the BjPIP1-overexpressing plants showed higher activities of antioxidative enzymes, and lower level of electrolyte leakage and malondialdehyde content under Cd stress, indicating BjPIP1 might enhance the antioxidative activity and membrane integrity in transgenic plants. Taken together, these results suggested that BjPIP1 might improve plant heavy-metal resistance through alleviating water deficit and oxidative damage induced by metal ions.