The outer membrane protein A (OmpA) of Yersinia pestis promotes intracellular survival and virulence in mice

The plague bacterium Yersinia pestis has a number of well-described strategies to protect itself from both host cells and soluble factors. In an effort to identify additional anti-host factors, we employed a transposon site hybridization (TraSH)-based approach to screen 10⁵Y. pestis mutants in an in vitro infection system. In addition to loci encoding various components of the well-characterized type III secretion system (T3SS), our screen unambiguously identified ompA as a pro-survival gene. We go on to show that an engineered Y. pestis ΔompA strain, as well as a ΔompA strain of the closely related pathogen Yersinia pseudotuberculosis, have fully functioning T3SSs but are specifically defective in surviving within macrophages. Additionally, the Y. pestis ΔompA strain was out competed by the wild-type strain in a mouse co-infection assay. Unlike in other bacterial pathogens in which OmpA can promote adherence, invasion, or serum resistance, the OmpA of Y. pestis is restricted to enhancing intracellular survival. Our data show that OmpA of the pathogenic Yersinia is a virulence factor on par with the T3SS.     

The C-terminus of IpaC is required for effector activities related to Shigella invasion of host cells

Invasion plasmid antigen C (IpaC) is secreted by the Shigella flexneri type III secretion system (TTSS) as an essential trigger of epithelial cell invasion. At the molecular level, IpaC possesses a distinct functional organization. The IpaC C-terminal region between amino acids 319 and 345 is predicted to form a coiled-coil structure. Such alpha-helical motifs appear to be a recurring structural theme among TTSS components. Together with IpaB, this IpaC region is also required for the formation of translocon pores in target cell membranes. In contrast, mutations within the C-terminal tail of IpaC (defined by residues 345-363) have no effect on contact hemolysis (a putative measure of translocon pore formation), but they can contribute significantly to IpaC's ability to trigger S. flexneri entry into cultured cells. Here we describe the molecular dissection of the IpaC C-terminus and how changes in this region affect selected virulence-related activities. IpaC invasion function requires its immediate C-terminus and this general region may be involved in its ability to trigger actin nucleation. In contrast, IpaC could not be shown to interact directly with Cdc42, a host GTPase closely tied to Shigella invasion.     

The roles of SaPI1 proteins gp7 (CpmA) and gp6 (CpmB) in capsid size determination and helper phage interference

SaPIs are molecular pirates that exploit helper bacteriophages for their own high frequency mobilization. One striking feature of helper exploitation by SaPIs is redirection of the phage capsid assembly pathway to produce smaller phage-like particles with T=4 icosahedral symmetry rather than T=7 bacteriophage capsids. Small capsids can accommodate the SaPI genome but not that of the helper phage, leading to interference with helper propagation. Previous studies identified two proteins encoded by the prototype element SaPI1, gp6 and gp7, in SaPI1 procapsids but not in mature SaPI1 particles. Dimers of gp6 form an internal scaffold, aiding fidelity of small capsid assembly. Here we show that both SaPI1 gp6 (CpmB) and gp7 (CpmA) are necessary and sufficient to direct small capsid formation. Surprisingly, failure to form small capsids did not restore wild-type levels of helper phage growth, suggesting an additional role for these SaPI1 proteins in phage interference.     

Co-administration of rIpaB domain of Shigella with rGroEL of S. Typhi enhances the immune responses and protective efficacy against Shigella infection

Shigella species cause severe bacillary dysentery in humans and are associated with high morbidity and mortality. The Invasion plasmid antigen (IpaB) protein, which is conserved across all Shigella spp., induces macrophage cell death and is required to invade host cells. The present study evaluates the immunogenicity and protective efficacy of the recombinant (r) domain region of IpaB (rIpaB) of S. flexneri. rIpaB was administered either alone or was co-administered with the rGroEL (heat shock protein 60) protein from S. Typhi as an adjuvant in a mouse model of intranasal immunization. The IpaB domain region (37 kDa) of S. flexneri was amplified from an invasion plasmid, cloned, expressed in BL21 Escherichia coli cells and purified. Immunization with the rIpaB domain alone stimulated both humoral and cell-mediated immune responses. Furthermore, robust antibody (IgG, IgA) and T-cell responses were induced when the rIpaB domain was co-administered with rGroEL. Antibody isotyping revealed higher IgG1 and IgG2a antibody titers and increased interferon-gamma (IFN-γ) secretion in the co-administered group. Immunization of mice with the rIpaB domain alone protected 60%–70% of the mice from lethal infection by S. flexneri, S. boydii and S. sonnei, whereas co-administration with rGroEL increased the protective efficacy to 80%–85%. Organ burden and histopathological studies also revealed a significant reduction in lung infection in the co-immunized mice compared with mice immunized with the rIpaB domain alone. This study emphasizes that the co-administration of the rIpaB domain and rGroEL protein improves immune responses in mice and increases protective efficacy against Shigella infection. This is also the first report to evaluate the potential of the GroEL (Hsp 60) protein of S. Typhi as an adjuvant molecule, thereby overcoming the need for commercial adjuvants.     

Localization of proteins associated with the outer hair cell plasma membrane in the gerbil cochlea

There is substantial evidence that the motility of mammalian outer hair cells is generated close to or within the plasma membrane. Several analogies between the outer hair cell cortical lattice and the membrane-related cytoskeleton of erythrocytes have been noted. In erythrocytes a member of the anion exchanger protein family, AE1, also known as Band 3, is involved in membrane-cytoskeleton linkage via Protein 4.1. In the following paper, the presence of these two proteins in gerbilline outer hair cells is confirmed by western blot. Furthermore, co-localization of these two proteins was detected in the lateral wall of outer hair cells by immunofluorescence and postembedding electron immunohistochemistry. Band 3 is restricted to this region, whereas Protein 4.1 has a somewhat more dispersed distribution.

Thus, the structure of these sensory receptor cells may result from an adaptation of a strategy used by other motile cells. The proteins investigated likely have a support function and may comprise “pillars” seen between the lateral plasma membrane and the cytoskeleton in micrographs of outer hair cells. The possibility that Band 3 comprises “protein particles” seen in the lateral plasma membrane, or maybe directly involved in the voltage-dependent force generation in outer hair cells, is also discussed.     

The multifunctional plant viral suppressor of gene silencing P19 interacts with itself and an RNA binding host protein

Tomato bushy stunt virus (TBSV) is an RNA plant virus encoding a protein of approximately 19 kDa (P19) that is involved in various activities important for pathogenicity, including virus transport and suppression of gene silencing. In this study, we provide evidence in vivo and in vitro that P19 specifically interacts with itself to predominantly form dimers, and with a novel host protein, Hin19. Hin19 has a high degree of similarity with a class of RNA-binding proteins of which many are involved in RNA processing. The binding of P19 to itself and to Hin19 both depend on a structurally important central region of P19 that was previously shown critical for its biological function in plants. Our findings provide evidence for a model in which virus spread through suppression of defense-related gene silencing involves the formation of a complex that includes P19 dimers and a newly identified host RNA-binding protein.     

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.     

Binding of outer membrane preparations of Campylobacter jejuni to INT 457 cell membranes and extracellular matrix proteins

Binding of outer membrane (OM) preparations of the thermophilic Campylobacter species C. jejuni to epithelial cell membranes and extracellular matrix proteins were studied in an in vitro model system using enzyme-linked immunosorbent assay. The OM preparations exhibited significant binding to INT 407 intestinal cell membranes. The process of adhesion was modulated by enzymatic, chemical or immunological pretreatment of the bacteria. Following oxidation of the lipopolysaccharide (LPS) with sodium meta-periodate, the OM preparations essentially retained their binding properties. After pretreatment with proteinase K, the OM preparations lost their binding capacity and the apparent molecular mass of the major OM protein shifted from 42 to 24 kDa. Preincubation of C. jejuni bacteria with C. jejuni-specific antiserum reduced adhesion significantly; preincubation with LPS-specific monoclonal antibodies only to a minimal extent. The OM preparations also bound significantly to the extracellular matrix proteins collagen and fibronectin; however, they bound virtually no bovine serum albumin or horse serum.     

Host recognition and integration of filamentous phage ?RSM in the phytopathogen, Ralstonia solanacearum

Two prophages, called ?RSM3 and ?RSM4, that are closely related to, but differ from, filamentous phage ?RSM1, have been detected in strains of the Ralstonia solanacearum species complex. The prophage ?RSM3, found in host strain MAFF730139, could be converted to infectious phage by means of PCR and transfection. The nucleotide sequence of ?RSM3 is highly conserved relative to ?RSM1 except for open reading frame 2 (ORF2), encoding an unknown protein, and ORF9 encoding the presumed adsorption protein that determines host range. The two host ranges differ dramatically and correlate closely with different gel electrophoresis banding patterns for cell surface fimbriae. Infections by ?RSM1 and ?RSM3 enhance bacterial cell aggregation and reduce the bacterial host virulence in tomato plants. Database searches in the R. solanacearum strains of known genomic sequence revealed two inovirus prophages, one designated ?RSM4 that is homologous to ?RSM1 and ?RSM3, and one homologues to RSS1, in the genome of strain UW551.     

Principal host relationships and evolutionary history of the North American arenaviruses

A previous study suggested that the genomes of the arenaviruses native to North America are a product of genetic recombination between New World arenaviruses with significantly different phylogenetic histories. The purpose of this study was to extend our knowledge of the principal host relationships and evolutionary history of the North American arenaviruses. The results of this study suggest that the large-eared woodrat (Neotoma macrotis) is a principal host of Bear Canyon virus and that the present-day association of Bear Canyon virus with the California mouse (Peromyscus californicus) in southern California represents a successful host-jumping event from the large-eared woodrat to the California mouse. Together, the results of analyses of viral gene sequence data in this study and our knowledge of the phylogeography of the rodents that serve as principal hosts of the New World arenaviruses suggest that genetic recombination between arenaviruses with significantly different phylogenetic histories did not play a role in the evolution of the North American arenaviruses.     

The mechanism of DNA ejection in the Bacillus anthracis spore-binding phage 8a revealed by cryo-electron tomography

The structure of the Bacillus anthracis spore-binding phage 8a was determined by cryo-electron tomography. The phage capsid forms a T = 16 icosahedron attached to a contractile tail via a head-tail connector protein. The tail consists of a six-start helical sheath surrounding a central tail tube, and a structurally novel baseplate at the distal end of the tail that recognizes and attaches to host cells. The parameters of the icosahedral capsid lattice and the helical tail sheath suggest protein folds for the capsid and tail-sheath proteins, respectively, and indicate evolutionary relationships to other dsDNA viruses. Analysis of 2518 intact phage particles show four distinct conformations that likely correspond to four sequential states of the DNA ejection process during infection. Comparison of the four observed conformations suggests a mechanism for DNA ejection, including the molecular basis underlying coordination of tail sheath contraction and genome release from the capsid.     

Transient cognitive deficits are associated with the reversible accumulation of amyloid precursor protein after mild traumatic brain injury

Mild traumatic brain injury (MTBI) may frequently cause transient behavioral abnormalities without observable morphological findings. In this study, we investigated neuropathological mechanisms underlying transient cognitive deficits after MTBI. Mongolian gerbils were subjected to experimental MTBI. At various time points after injury, behavioral changes were evaluated by the open-field test and T-maze test, and immunohistochemistry of microtubule-associated protein (MAP2) and amyloid precursor protein (APP) was performed to examine disruptions of the neuronal cytoskeleton and axonal transport, respectively. Transient cognitive deficits were observed after MTBI. Sustained MAP2 loss was found within the cortical impact site, but not the hippocampus. Transient APP accumulation at the same time as transient cognitive deficits occurred in the ipsilateral hemisphere, particularly in the subcortical white matter. These results suggest that the axonal dysfunction indicated by the reversible APP accumulation in the white matter, but not the sustained neuronal cytoskeletal damage reflected by the cortical MAP2 loss confined to the impact site, is responsible for the transient functional deficits after MTBI.     

The Sulfolobus rod-shaped virus 2 encodes a prominent structural component of the unique virion release system in Archaea

Recently a unique mechanism of virion release was discovered in Archaea, different from lysis and egress systems of bacterial and eukaryotic viruses. It involves formation of pyramidal structures on the host cell surface that rupture the S-layer and by opening outwards, create apertures through which mature virions escape the cell. Here we present results of a protein analysis of Sulfolobus islandicus cells infected with the rudivirus SIRV2, which enable us to postulate SIRV2-encoded protein P98 as the major constituent of these exceptional cellular ultrastructures.     

Polymorphisms in LMNA and near a SERPINA13 gene are not associated with cognitive performance in Chinese elderly males without dementia

Aging is associated with cognitive deterioration. A recent study showed two polymorphisms (rs505058 in LMNA and rs11622883 near a SERPINA13 gene), identified in a genome-wide association study of late-onset Alzheimer's disease, to be associated with cognitive function (Mini Mental State Examination) in a UK elderly population. This study replicated these findings in Chinese elderly males without dementia. A total of 358 elderly subjects were assessed by the Cognitive Abilities Screening Instruments (CASI) and the Wechsler Digit Span Task tests. Analysis of covariance was used to compare cognitive scores among genotypic groups, with age and total education years as covariates. The two polymorphisms were not associated with the global cognitive function or specific cognitive domains in the elderly without dementia. Our data argue against that these two polymorphisms may affect cognitive function in the elderly.     

Role of the hemin-binding protein 35 (HBP35) of Porphyromonas gingivalis in coaggregation

Hemin-binding protein 35 (HBP35) in Porphyromonas gingivalis is one of the outer membrane proteins and has been reported to be a non-fimbrial coaggregation factor. In this study, a P. gingivalis HBP35-deficient mutant (MD774) was constructed from wild-type strain FDC381 by insertion mutagenesis in order to provide a better understanding of this protein's role in coaggregation. The intact cells and vesicles in FDC381 were found to have strong aggregation activities with Gram-positive bacteria. But neither the vesicles nor the intact cells showed aggregation activity in MD774. In addition, MD774 reduced autoaggregation activity. Immunoblot analysis of MD774 showed the presence of a non-maturated 45-kDa fimbrillin protein. Electron microscopy showed that the MD774 had no long fimbriae on the cell surface. Arg- and Lys-gingipain activity in MD774 was significantly decreased, compared with FDC381. Real-time RT-PCR demonstrated a significant reduction in the expression of gingipain-associated genes rgpA, rgpB, and kgp. In conclusion, we suggest that the reduction in coaggregation was caused by the combined reduction of a variety of molecules, including HBP35, gingipains, and fimbriae. Our results suggest that the HBP35 protein directly influences not only coaggregation as an adhesion molecule but also indirectly influences the expression of other coaggregation factors.     

Trypanosoma cruzi heparin-binding proteins and the nature of the host cell heparan sulfate-binding domain

Trypanosoma cruzi invasion is mediated by receptor-ligand recognition between the surfaces of both parasite and target cell. We have previously demonstrated the role of heparan sulfate proteoglycan in the attachment and invasion of T. cruzi in cardiomyocytes. Herein, we have isolated the T. cruzi heparin-binding proteins (HBP-Tc) and investigated the nature of cardiomyocyte heparan sulfate (HS)-binding site to the parasite surface ligand. Two major heparin-binding proteins with molecular masses of 65.8 and 59 kDa were observed in total extract of amastigote and trypomastigote forms of T. cruzi. Hydrophobic [S(35)]methionine labeled proteins eluted from heparin-sepharose affinity chromatography also revealed both proteins in trypomastigotes but only the 59 kDa is strongly recognized by biotin-conjugated glycosaminoglycans. Competition assays were performed to analyze the role of sulfated proteoglycans, including heparin, keratan sulfate and both acetylated and highly sulfated domains of heparan sulfate, in the recognition and invasion process of T. cruzi. Significant inhibitions of 84% and 35% in the percentage of infection were revealed after treatment of the parasites with heparin and the N-acetylated/ N-sulfated heparan sulfate domain, respectively, suggesting the important role of the glycuronic acid and NS glucosamine domain of the HS chain in the recognition of the HBP-Tc during the T. cruzi-cardiomyocyte interaction.     

Cymbidium mosaic potexvirus isolate-dependent host movement systems reveal two movement control determinants and the coat protein is the dominant

Little is known about how plant viruses of a single species exhibit different movement behavior in different host species. Two Cymbidium mosaic potexvirus (CymMV) isolates, M1 and M2, were studied. Both can infect Phalaenopsis orchids, but only M1 can systemically infect Nicotiana benthamiana plants. Protoplast inoculation and whole-mount in situ hybridization revealed that both isolates can replicate in N. benthamiana; however, M2 was restricted to the initially infected cells. Genome shuffling between M1 and M2 revealed that two control modes are involved in CymMV host dependent movement. The M1 coat protein (CP) plays a dominant role in controlling CymMV movement between cells, because all chimeric CymMV viruses containing the M1 CP systemically infected N. benthamiana plants. Without the M1 CP, one chimeric virus containing the combination of the M1 triple gene block proteins (TGBps), the M2 5′ RNA (1-4333), and the M2 CP effectively moved in N. benthamiana plants. Further complementation analysis revealed that M1 TGBp1 and TGBp3 are co-required to complement the movement of the chimeric viruses in N. benthamiana. The amino acids within the CP, TGBp1 and TGBp3 which are required or important for CymMV M2 movement in N. benthamiana plants were mapped. The required amino acids within the CP map to the predicted RNA binding domain. RNA-protein binding assays revealed that M1 CP has higher RNA binding affinity than does M2 CP. Yeast two-hybrid assays to detect all possible interactions of M1 TGBps and CP, and only TGBp1 and CP self-interactions were observed.     

Outer membrane protein A expression in Enterobacter sakazakii is required to induce microtubule condensation in human brain microvascular endothelial cells for invasion

Enterobacter sakazakii (ES) causes neonatal meningitis and necrotizing enterocolitis with case-fatality rates among infected infants ranging from 40 to 80%. Very little is known about the mechanisms by which these organisms cause disease. Here, we demonstrate that ES invades human brain microvascular endothelial cells (HBMEC) with higher frequency when compared with epithelial cells and endothelial cells from different origins. The entry of ES into HBMEC requires the expression of outer membrane protein A (OmpA), as the OmpA-deletion mutant was sevenfold less invasive than the wild type ES and the bacterium does not multiply inside HBMEC. Anti-OmpA antibodies generated against the OmpA of Escherichia coli K1, which also recognize the OmpA of ES, did not prevent the invasion of ES in HBMEC. ES invasion depends on microtubule condensation in HBMEC and is independent of actin filament reorganization. Both PI3-kinase and PKC-alpha were activated during ES entry into HBMEC between 15min and 30min of infection. Concomitantly, overexpression of dominant negative forms of PI3-kinase and PKC-alpha significantly inhibited the invasion of ES into HBMEC. In summary, ES invasion of HBMEC is dependent on the expression of OmpA similar to that of E. coli K1; however, the epitopes involved in the interaction with HBMEC appears to be different.     

Identification and characterization of Inc766, an inclusion membrane protein in Chlamydophila abortus-infected cells

We have identified the gene product of locus 766 in the transmembrane head region (TMH/Inc-region) in the Chlamydophila abortus genome by using mass spectrometry and a monoclonal antibody that reacted with the inclusion membrane. The identified protein at 32kDa, termed Inc766, formed highly stable oligomers when solubilized in the absence of beta-mercaptoethanol. These oligomers were resistant to SDS, to heat denaturation and to 8M urea, but very sensitive to beta-mercaptoethanol, consistent with conformations resulting from protein-protein interactions stabilized through disulphide bonds. Mass spectrometry analysis of immunoprecipitated infected cell lysates indicated that a dimer at 56kDa was the most prominent form in solution. Cross-linking with DSP provided supporting evidence for the formation of oligomers in situ. Inc766 was expressed at 20-24h post infection and its localization pattern in the extra-inclusion space was common in all C. abortus strains tested. Taken together, Inc766 displays unique biochemical and cellular features not encountered in other Incs from other Chlamydiaceae species. Future studies of the particular characteristics especially the interactive properties of Inc766 should contribute to our understanding of the relationship of the different chlamydial species with their respective hosts.