P087, a lactococcal phage with a morphogenesis module similar to an Enterococcus faecalis prophage

The virulent lactococcal phage P087 was isolated from a dairy environment in 1978. This phage was then recognized as the reference member for one of the ten phage groups currently known to infect Lactococcus lactis strains. The double-stranded DNA genome of this Siphoviridae phage is composed of 60,074 bp and is circularly permuted. Five tRNA and 88 orfs were found within an uncommon genome architecture. Eleven structural proteins were also identified through SDS-PAGE and LC-MS/MS analyses. Of note, 11 translated orfs from the structural module of phage P087 have identities to gene products found in a prophage located in the genome of Enterococcus faecalis V583. The alignment of both genomic sequences suggests that DNA exchanges could occur between these two phages which are infecting low G+C bacteria found in similar ecological niches.     

Role of recombination in the evolution of natural populations of Cucumber mosaic virus, a tripartite RNA plant virus

The role of recombination in the evolution of Cucumber mosaic virus (CMV) was analyzed in a collection of Spanish isolates from 1989 to 2002. Isolates were characterized by ribonuclease protection assay using six RNA probes, two for each of the three genomic RNAs, which allowed the identification of the analyzed regions as belonging to CMV isolates in subgroups IA, IB, and II. Most isolates belonged to subgroups IA (64%) and IB (12%), 5% were reassortants among subgroups IA, IB, or II, and 17% were recombinants between these groups. Recombinants at RNA3 were significantly more frequent than recombinants at RNAs 1 and 2. One IB-IA recombinant RNA3 was as frequent in central Spain as the IA RNA3. The genetic structure of the virus population suggested that reassortants and most recombinant genotypes were selected against and was consistent with a higher biological cost of reassortment than recombination. Data also suggest that recombinants that encode hybrid proteins are at a higher disadvantage than recombinants that exchange whole ORFs.     

Anti-pneumococcal deficits of monocyte-derived macrophages from the advanced-age,frail elderly and related impairments in PI3K-AKT signaling

The advanced-age, frail elderly are especially vulnerable to developing pneumococcal infection and disease. Macrophages are critical mediators in the defence against Streptococcus pneumoniae at the upper respiratory tract, however, little is known of their anti-pneumococcal capacity in the elderly. Herein we demonstrate that monocyte-derived macrophages (MDMs) from the advanced-age, frail elderly produce less TNF, IL-6, IL-1β and IL-8 in response to heat-killed S. pneumoniae, which does not appear to be related to mRNA stability or decay. Furthermore, despite maintaining the ability to bind and phagocytose bacteria, MDMs from these individuals have a reduced capacity to kill S. pneumoniae. These defects parallel reduced PI3K-AKT signaling, which can significantly abrogate bacterial killing, but does not affect cytokine responses. Since macrophages are critical in the defence against S. pneumoniae, this study adds valuable insight into the susceptibility of the elderly to pneumococcal disease and highlights the PI3K-AKT signaling pathway as a potential therapeutic target.     

Genomic organization and mutation screening of the human ortholog of Pkdr1 associated with polycystic kidney disease in the rat

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited disorders in humans. Although disease-causing mutations have been found in two genes, PKD1 and PKD2, a small number of ADPKD families exist that are unlinked to either of these genes, suggesting involvement of a third, as yet unidentified PKD3 gene. Susceptibility to renal cyst formation in the (cy/+) rat is caused by a missense mutation in Pkdr1 encoding the novel protein SamCystin. To initiate studies of the human orthologous gene, we determined the location and the organization of human PKDR1. We genotyped microsatellite markers flanking the human ortholog in PKD families that either are unlinked to known PKD genes, or in which mutations have not yet been identified and carried out mutation analysis in PKD patients. We identified eight novel single nucleotide polymorphisms, including three leading to amino acid changes. These variants are unlikely to account for PKD in these patients, yet the screening of other affected populations may provide information about the involvement of PKDR1 as a modifier gene in cystic kidney disease.     

Synergism of a DNA and an RNA virus: enhanced tissue infiltration of the begomovirus Abutilon mosaic virus (AbMV) mediated by Cucumber mosaic virus (CMV)

Replication of the begomovirus Abutilon mosaic virus (AbMV) is restricted to phloem nuclei, generating moderate levels of virus DNA. Co-infection with Cucumber mosaic virus (CMV) evidently increased AbMV titers in Nicotiana benthamiana, tobacco, and tomato, resulting in synergistic symptom enhancement. In situ hybridization revealed that in double-infected leaves an increased number of nuclei contained elevated amounts of AbMV. Additionally, the begomoviral phloem-limitation was broken. Whereas CMV 3a movement protein-expressing tobacco plants did not exert any similar influence, the presence of CMV 2b silencing suppressor protein lead to enhanced AbMV titers and numbers of infected vascular cells. The findings prove that AbMV can replicate in nonvascular cells and represent the first report on a true synergism of an RNA/ssDNA virus combination in plants, in which CMV 2b protein plays a role. They indicate considerable consequences of mixed infections between begomo- and cucumoviruses on virus epidemiology and agriculture.     

Cell-to-cell movement of Alfalfa mosaic virus can be mediated by the movement proteins of Ilar-, bromo-, cucumo-, tobamo- and comoviruses and does not require virion formation

RNA 3 of Alfalfa mosaic virus (AMV) encodes the movement protein (MP) and coat protein (CP). Chimeric RNA 3 with the AMV MP gene replaced by the corresponding MP gene of Prunus necrotic ringspot virus, Brome mosaic virus, Cucumber mosaic virus or Cowpea mosaic virus efficiently moved from cell-to-cell only when the expressed MP was extended at its C-terminus with the C-terminal 44 amino acids of AMV MP. MP of Tobacco mosaic virus supported the movement of the chimeric RNA 3 whether or not the MP was extended with the C-terminal AMV MP sequence. The replacement of the CP gene in RNA 3 by a mutant gene encoding a CP defective in virion formation did not affect cell-to-cell transport of the chimera's with a functional MP. A GST pull-down technique was used to demonstrate for the first time that the C-terminal 44 amino acids of the MP of a virus belonging to the family Bromoviridae interact specifically with AMV virus particles. Together, these results demonstrate that AMV RNA 3 can be transported from cell-to-cell by both tubule-forming and non-tubule-forming MPs if a specific MP-CP interaction occurs.     

Early Changes in the Serotype Distribution of Invasive Pneumococcal Isolates from Children after the Introduction of Extended-valent Pneumococcal Conjugate Vaccines in Korea, 2011-2013

This study was performed to measure early changes in the serotype distribution of pneumococci isolated from children with invasive disease during the 3-year period following the introduction of 10- and 13-valent pneumococcal conjugate vaccines (PCVs) in Korea. From January 2011 to December 2013 at 25 hospitals located throughout Korea, pneumococci were isolated among children who had invasive pneumococcal disease (IPD). Serotypes were determined using the Quellung reaction, and the change in serotype distribution was analyzed. Seventy-five cases of IPD were included. Eighty percent of patients were aged 3-59 months, and 32% had a comorbidity that increased the risk of pneumococcal infection. The most common serotypes were 19A (32.0%), 10A (8.0%), and 15C (6.7%). The PCV7 serotypes (4, 6B, 9V, 14, 18C, 19F, 23F, and 6A) accounted for 14.7% of the total isolates and the PCV13 minus PCV7 types (1, 3, 5, 7F and 19A) accounted for 32.0% of the total isolates. Serotype 19A was the only serotype in the PCV13 minus PCV7 group. The proportion of serotype 19A showed decreasing tendency from 37.5% in 2011 to 22.2% in 2013 (P = 0.309), while the proportion of non-PCV13 types showed increasing tendency from 45.8% in 2011 to 72.2% in 2013 (P = 0.108). Shortly after the introduction of extended-valent PCVs in Korea, serotype 19A continued to be the most common serotype causing IPD in children. Subsequently, the proportion of 19A decreased, and non-vaccine serotypes emerged as an important cause of IPD. The impact of extended-valent vaccines must be continuously monitored.     

Spatial analysis for exclusive interactions between subgroups I and II of Cucumber mosaic virus in cowpea

The dynamics of virus interference in Cucumber mosaic virus (CMV) infection in cowpea were investigated by tissue-blotting and in situ hybridization. Using co-inoculation assays, we discovered that spatial competition between CMV-LE (subgroup I) and CMV-m2 (subgroup II) occurred in the inoculated leaves. Interestingly, competitive interactions between the two viruses also could be observed in the non-inoculated upper leaf tissues of the plants. Furthermore, the pattern of exclusive distribution was observed between challenge and protecting viruses in the serially inoculated leaves. Taken together, it is suggested that the dynamics of competitive interactions between the two subgroups could be characterized by exclusive infection and multiplication of the individual viruses in cowpea plants.     

Characterization of 1706, a virulent phage from Lactococcus lactis with similarities to prophages from other Firmicutes

The virulent lactococcal phage 1706, isolated in 1995 from a failed cheese production in France, represents a new lactococcal phage species of the Siphoviridae family. This phage has a burst size of 160 and a latent period of 85 min. Its linear double-stranded DNA genome was composed of 55,597 bp with a 33.7% G+C content. Its deduced proteome (76 ORFs) shared limited similarities to other known phage proteins. SDS-PAGE coupled with LC-MS/MS analyses led to the identification of 15 structural proteins. The most striking feature of the 1706 proteome was that 22 ORFs shared similarities with proteins deduced from the genome of either Ruminococcus torques and/or Clostridium leptum. Both are Firmicutes bacteria found in the gut flora of humans. We also identified a four-gene module in phage 1706, most likely involved in host recognition that shared similarities with lactococcal prophages. We propose that the virulent phage 1706 infected another bacterial genus before picking up a lactococcal host recognition module.     

Serotype-specific penicillin resistance of Streptococcus pneumoniae in Germany from 1992 to 2008

A total of 12,137 isolates from invasive pneumococcal disease was collected between January 1, 1992, and December 31, 2008, by the German National Reference Center for Streptococci (NRCS). Data on penicillin susceptibility were available for 11,814 isolates, whereat 8837 isolates (74.8%) were from adults, and 2977 isolates (25.2%) originated from children. Overall, the leading serotypes were serotypes 14 (16.5% of serotyped isolates), 3 (8.1%), 7F (7.7%), 1 (7.4%), and 23F (6.0%). The overall nonsusceptibility rate of all isolates adds up to 5.5% (intermediate, 4.3%; resistant, 1.2%) when the CLSI 2006 guidelines were applied, and to 1.4% (intermediate, 0.2%; resistant, 1.2%) when using the CLSI 2009 guidelines. Generally, slightly higher resistance rates were observed among children than among adults. Serotypes contributing considerably to pneumococcal penicillin nonsusceptibility by a combination of frequency among invasive isolates and relatively high penicillin nonsusceptibility are 19A, 9V, 6B, 19F, 23F, and 14. While the nonsusceptibility among serotype 19A isolates increased considerably over the years, the development of nonsusceptibility rates among the other serotypes is less and more ambiguous.     

The interaction between bacterial enolase and plasminogen promotes adherence of Streptococcus pneumoniae to epithelial and endothelial cells

Binding and conversion of the plasma protein plasminogen is an important pathogenesis mechanism of the human pathogen Streptococcus pneumoniae. Once converted into plasmin, the proteolytic activity of this major fibrinolysis component promotes degradation of extracellular matrix and the dissolution of fibrin clots. Here, we present the exploitation of plasminogen-binding as a further pivotal strategy of pneumococci facilitating adherence to eukaryotic cells. Flow cytometric measurements demonstrated the immobilization of plasminogen on host cell surfaces of human alveolar type II pneumocytes (A549), nasopharyngeal epithelium (Detroit 562) and brain-derived endothelial cells (HBMEC). These host-derived cells were employed in cell culture infection analyses followed by confocal microscopy to monitor the plasminogen-mediated adherence. Results of these studies revealed that host cell-bound plasminogen promotes pneumococcal adherence to human epithelial and endothelial cells in dose-dependent manner, whereas pneumococcal internalization was not enhanced. As an opposed effect pneumococcal-bound plasminogen reduced attachment to the epithelial and endothelial cells, and increased the interaction with neutrophil granulocytes. Moreover, the surface-displayed enolase, which serves as major pneumococcal plasminogen receptor, was identified as a key factor for plasminogen-mediated bacterial attachment in infection analyses with S. pneumoniae enolase mutants.     

Efficient and stable expression of GFP through Wheat streak mosaic virus-based vectors in cereal hosts using a range of cleavage sites: formation of dense fluorescent aggregates for sensitive virus tracking

A series of Wheat streak mosaic virus (WSMV)-based expression vectors were developed by engineering a cycle 3 GFP (GFP) cistron between P1 and HC-Pro cistrons with several catalytic/cleavage peptides at the C-terminus of GFP. WSMV-GFP vectors with the Foot-and-mouth disease virus 1D/2A or 2A catalytic peptides cleaved GFP from HC-Pro but expressed GFP inefficiently. WSMV-GFP vectors with homologous NIa-Pro heptapeptide cleavage sites did not release GFP from HC-Pro, but efficiently expressed GFP as dense fluorescent aggregates. However, insertion of one or two spacer amino acids on either side of NIb/CP heptapeptide cleavage site or deletion in HC-Pro cistron improved processing by NIa-Pro. WSMV-GFP vectors were remarkably stable in wheat for seven serial passages and for 120 days postinoculation. Mite transmission efficiencies of WSMV-GFP vectors correlated with the amount of free GFP produced. WSMV-GFP vectors infected the same range of cereal hosts as wild-type virus, and GFP fluorescence was detected in most wheat tissues.     

Changes in serotype distribution and antibiotic resistance of nasopharyngeal isolates of Streptococcus pneumoniae from children in Korea, after optional use of the 7-valent conjugate vaccine

We investigated serotype distribution and antimicrobial resistance of pneumococcal carriage isolates from children after optional immunization with the 7-valent pneumococcal conjugate vaccine (PCV7) in Korea. From June 2009 to June 2010, 205 (16.5%) pneumococcal isolates were obtained from 1,243 nasopharyngeal aspirates of infants and children at Seoul National University Children's Hospital, Korea. Serotype was determined by Quellung reaction and antibiotic susceptibility was tested by E-test. The results were compared to previous studies done in the pre-PCV7 period. In this study, the most common serotypes were 6A (15.3%), 19A (14.7%), 19F (10.2%), 35B (7.3%), and 6D (5.6%). The proportion of PCV7 serotypes decreased from 61.9% to 23.8% (P < 0.001). The overall penicillin nonsusceptibility rate increased from 83.5% to 95.4% (P = 0.001). This study demonstrates the impact of optional PCV7 vaccination in Korea; the proportion of all PCV7 serotypes except 19F decreased while antimicrobial resistant serotypes 6A and 19A further increased.     

Mutation analysis of the parkin and PINK1 genes in American Caucasian early-onset Parkinson disease families

Mutations in the parkin gene and the PTEN-induced putative kinase 1 gene (PINK1) have been identified as the most common causes of autosomal recessive early-onset Parkinson disease (EOPD). To investigate the presence of the parkin and PINK1 gene mutation(s) and to explore genotype-phenotype correlations in American Caucasian families with EOPD from North American, we screened these two genes in probands of six families by direct sequencing, semi-quantitative PCR and RT-PCR. No PINK1 gene mutation was found in any of the probands, but compound heterozygous mutations (EX 3 del and EX 3_4 del) in the parkin gene were identified in one family. Extended analysis of the parkin-positive family showed the phenotype of patients was that of classic autosomal recessive EOPD, characterized by early age at onset, slow progression, beneficial response to levodopa, and levodopa-related motor complications. Three heterozygous mutation carriers (EX 3 del or EX 3_4 del) were free of any neurological symptoms. None of 62 healthy controls harbored EX 3 del or EX 3_4 del mutation. Our data suggest that compound heterozygous mutations (EX 3 and EX 3_4 del) in the parkin gene were the cause of EOPD in one of six Caucasian families; heterozygous EX 3 del and heterozygous EX 3_4 del forms were insufficient to cause this disorder, consistent with a loss-of-function mechanism of the parkin mutations. The results may provide new insights into the cause and diagnosis of PD and have implications for genetic counseling.     

Morphology of temperate bacteriophage SfV and characterisation of the DNA packaging and capsid genes: the structural genes evolved from two different phage families

The entire genome of SfV, a temperate serotype-converting bacteriophage of Shigella flexneri, has recently been sequenced (Allison, G.E., Angeles, D., Tran-Dinh, N., Verma, N.K. 2002, J. Bacteriol. 184, 1974-1987). Based on the sequence analysis, we further characterised the SfV virion structure and morphogenesis. Electron microscopy indicated that SfV belongs to the Myoviridae morphology family. Analysis of the proteins encoded by orf1, orf2, and orf3 revealed that they were homologous to small and large terminase subunits, and portal proteins, respectively; the protein encoded by orf5 showed homology to capsid proteins. Western immunoblot of the phage with anti-SfV sera revealed two antigenic proteins, and the N-terminal amino acid sequence of the 32-kDa protein corresponded to amino acids 116 to 125 of the ORF5 protein, suggesting that the capsid may be processed. Functional analysis of orf4 showed that it encodes the phage capsid protease. The proteins encoded by orfs1, 2, 3, 4, and 5 are homologous to similar proteins in the Siphoviridae phage family of both gram-positive and gram-negative origin. The capsid and morphogenesis genes are upstream and adjacent to the genes encoding Myoviridae (Mu-like) tail proteins. The organisation of the structural genes of SfV is therefore unique as the head and tail genes originate from different morphology groups.     

Molecular mechanisms of the secretion of cytokines and chemokines from human monocytes activated by pneumococcal surface protein A (PspA): Roles of mitogen-activated protein kinases and NF-kappaB

Pneumococcal surface protein A (PspA) plays a key role in the pathogenesis of invasive pneumococcal infection. PspA might modulate specific immune responses in human population. Circulating monocytes are essential for the innate responses and subsequent acquired immune responses to Streptococcus pneumoniae. In this study, we investigated the effects of PspA on cytokine and chemokine secretion from human peripheral blood monocytes and the underlying intracellular signaling mechanisms. Stimulation of monocytes with purified PspA protein induced the significant release of inflammatory cytokine IL-6 and chemokines including CXCL8, CCL2, CCL4 and CCL5. Products from PspA-deficient mutant pneumococcus that did not express PspA induced significantly less secretion of these mediators than those from wild type pneumococcus. Further investigations showed that PspA activated the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen activated protein kinase (MAPK) and nuclear factor (NF)-κB signaling pathways in human monocytes. Moreover, inhibition of these pathways using selective inhibitors could significantly reduce the cytokine and chemokine secretion induced by PspA. Taken together, our findings provide insight for PspA-mediated activation of human monocytes via NF-κB and MAPKs signaling cascades in the pathogenesis of invasive pneumococcal infection.