A nine-base nucleotide sequence in the porcine circovirus type 2 (PCV2) nucleocapsid gene determines viral replication and virulence

Porcine circovirus type 2 (PCV2) was retrospectively identified by serology in swine populations as an asymptomatic infection at least 25 years prior to the first reported case of PCV2-associated postweaning multisystemit wasting syndrome (PMWS). To investigate the sudden emergence of PMWS, viral sequences were amplified from frozen archived (1970-1971) porcine tissues and the complete genome of archival PCV2 was determined. The ORF1 gene product (viral DNA replicase) was homologous to contemporary PCV2 ORF1. In ORF2 (viral nucleocapsid gene) archival PCV2, a consistent linear nine-base sequence difference at base positions 1331 through 1339 was observed.The deduced amino acid sequence from these base changes alters the nucleocapsid conformation within the second immunogenic epitope from a hydrophobic (contemporary PCV2) to a hydrophilic (archival PCV2) configuration.

Detection of a novel porcine boca-like virus in the background of porcine circovirus type 2 induced postweaning multisystemic wasting syndrome

Porcine circovirus type 2 (PCV-2) has been found to be the causative agent of postweaning multisystemic wasting syndrome (PMWS). However, PCV-2 is a ubiquitous virus in the swine population and a majority of pigs infected with PCV-2 do not develop the disease. Different factors such as age, maintenance, the genetics of PCV-2, other pathogens, etc. have been suggested to contribute to the development of PMWS. However, so far no proven connection between any of these factors and the disease development has been found. In this study we explored the possible presence of other so far unknown DNA containing infectious agents in lymph nodes collected from Swedish pigs with confirmed PMWS through random amplification and high-throughput sequencing. Although the vast majority of the amplified genetic sequences belonged to PCV-2, we also found genome sequences of Torque Teno virus (TTV) and of a novel parvovirus. The detection of TTV was expected since like PCV-2, TTV has been found to have high prevalence in pigs around the world. We were able to amplify a longer region of the parvovirus genome, consisting of the entire NP1 and partial VP1/2. By comparative analysis of the nucleotide sequences and phylogenetic studies we propose that this is a novel porcine parvovirus, with genetic relationship to bocaviruses.

Bacteriophages as anti-infective agents: recent developments and regulatory challenges.

The biennial meeting on 'Exploiting Bacteriophages for Bioscience, Biotechnology and Medicine', held in London, UK, on 20 January 2012, and chaired by George Salmond (University of Cambridge, UK) hosted over 50 participants representing 13 countries. The highly multidisciplinary meeting covered a diverse range of topics, reflecting the current expansion of interest in this field, including the use of bacteriophages as the source of biochemical reagents for molecular biology, bacteriophages for the treatment of human and animal diseases, bacteriophage-based diagnostics and therapeutic delivery technologies and necessity for, and regulatory challenges associated with, robust clinical trials of phage-based therapeutics. This report focuses on a number of presentations from the meeting relating to cutting-edge research on bacteriophages as anti-infective agents.

Synergistic phage-antibiotic combinations for the control of Escherichia coli biofilms in vitro.

The potential application of phage therapy for the control of bacterial biofilms has received increasing attention as resistance to conventional antibiotic agents continues to increase. The present study identifies antimicrobial synergy between bacteriophage T4 and a conventional antibiotic, cefotaxime, via standard plaque assay and, importantly, in the in vitro eradication of biofilms of the T4 host strain Escherichia coli 11303. Phage-antibiotic synergy (PAS) is defined as the phenomenon whereby sub-lethal concentrations of certain antibiotics can substantially stimulate the host bacteria's production of virulent phage. Increasing sub-lethal concentrations of cefotaxime resulted in an observed increase in T4 plaque size and T4 concentration. The application of PAS to the T4 one-step growth curve also resulted in an increased burst size and reduced latent period. Combinations of T4 bacteriophage and cefotaxime significantly enhanced the eradication of bacterial biofilms when compared to treatment with cefotaxime alone. The addition of medium (10(4) PFU mL(-1) ) and high (10(7) PFU mL(-1) ) phage titres reduced the minimum biofilm eradication concentration value of cefotaxime against E. coli ATCC 11303 biofilms from 256 to 128 and 32 µg mL(-1) , respectively. Although further investigation is needed to confirm PAS, this study demonstrates, for the first time, that synergy between bacteriophage and conventional antibiotics can significantly improve biofilm control in vitro.

Type IVB pili are required for invasion but not for adhesion of Salmonella enterica serovar Typhi into BHK epithelial cells in a cystic fibrosis transmembrane conductance regulator-independent manner

The cystic fibrosis transmembrane conductance regulator (CFTR) has been proposed as an epithelial cell receptor for the entry of Salmonella Typhi but not Salmonella Typhimurium. The bacterial ligand recognized by CM is thought to reside either in the S. Typhi lipopolysaccharide core region or in the type IV pili. Here, we assessed the ability of virulent strains of S. Typhi and S. Typhimurium to adhere to and invade BHK epithelial cells expressing either the wild-type CFTR protein or the Delta F508 CFTR mutant. Both S. Typhi and S. Typhimurium invaded the epithelial cells in a CFTR-independent fashion. Furthermore and also in a CFTR-independent manner, a S. Typhi pilS mutant adhered normally to BHK cells but displayed a 50% reduction in invasion as compared to wild-type bacteria. Immunofluorescence microscopy revealed that bacteria and CFTR do not colocalize at the epithelial cell surface. Together, our results strongly argue against the established dogma that CFTR is a receptor for entry of Salmonella to epithelial cells. (C) 2011 Elsevier Ltd. All rights reserved.

Different sugar residues of the lipopolysaccharide outer core are required for early interactions of Salmonella enterica serovars Typhi and Typhimurium with epithelial cells

The role of lipopolysaccharide (LPS) in entry of Salmonella Typhimurium into epithelial cells remains unclear. In this study, we tested the ability of a series of mutants with deletions in genes for the synthesis and assembly of the O antigen and the outer core of LPS to adhere to and invade HeLa, BHK, and IB3 epithelial cells lines. Mutants devoid of O antigen, or that synthesized only one O antigen unit, or with altered O antigen chain lengths were as able as the wild type to enter epithelial cells, indicating that this polysaccharide is not required for invasion of epithelial cells in vitro. In contrast, the LPS core plays a role in the interaction of S. Typhimurium with epithelial cells. The minimal core structure required for adherence and invasion comprised the inner core and residues Glc I Gal I of the outer core. A mutant of S. Typhimurium that produced a truncated LPS core lacking the terminal galactose residue had a significant lower level of adherence to and ingestion by the three epithelial cell lines than did strains with this characteristic. Complementation of the LPS production defect recovered invasion to parental levels. Heat-killed bacteria with a core composed of Glc 1 Gal I. but not bacteria with a core composed of Glc 1, inhibited uptake of the wild type by HeLa cells. A comparison of the chemical structure of the S. Typhi core with the published chemical structure of that of S. Typhimurium indicated that the Glc I Gal 1 Glc 11 backbone is conserved in both serovars. However, S. Typhi requires a terminal glucose for maximal invasion. Therefore, our data indicate that critical saccharide residues of the outer core play different roles in the early interactions of serovars Typhi and Typhimurium with epithelial cells. (C) 2010 Elsevier Ltd. All rights reserved.

A Decade of Burkholderia cenocepacia Virulence Determinant Research

The Burkholderia cepacia complex (Bcc) is a group of genetically related environmental bacteria that can cause chronic opportunistic infections in patients with cystic fibrosis (CF) and other underlying diseases. These infections are difficult to treat due to the inherent resistance of the bacteria to antibiotics. Bacteria can spread between CF patients through social contact and sometimes cause cepacia syndrome, a fatal pneumonia accompanied by septicemia. Burkholderia cenocepacia has been the focus of attention because initially it was the most common Bcc species isolated from patients with CF in North America and Europe. Today, B. cenocepacia, along with Burkholderia multivorans, is the most prevalent Bcc species in patients with CF. Given the progress that has been made in our understanding of B. cenocepacia over the past decade, we thought that it was an appropriate time to review our knowledge of the pathogenesis of B. cenocepacia, paying particular attention to the characterization of virulence determinants and the new tools that have been developed to study them. A common theme emerging from these studies is that B. cenocepacia establishes chronic infections in immuno-compromised patients, which depend more on determinants mediating host niche adaptation than those involved directly in host cells and tissue damage.

An anthrax subunit vaccine candidate based on protective regions of Bacillus anthracis protective antigen and lethal factor

Studies have confirmed the key role of Bacillus anthracis protective antigen (PA) in the US and UK human anthrax vaccines. However, given the tripartite nature of the toxin, other components, including lethal factor (LF), are also likely to contribute to protection. We examined the antibody and T cell responses to PA and LF in human volunteers immunized with the UK anthrax vaccine (AVP). Individual LF domains were assessed for immunogenicity in mice when given alone or with PA. Based on the results obtained, a novel fusion protein comprising D1 of LF and the host cell-binding domain of PA (D4) was assessed for protective efficacy. Murine protection studies demonstrated that both full-length LF and D1 of LF conferred complete protection against a lethal intraperitoneal challenge with B. anthracis STI spores. Subsequent studies with the LFD1-PAD4 fusion protein showed a similar level of protection. LF is immunogenic in humans and is likely to contribute to the protection stimulated by AVP. A single vaccine comprising protective regions from LF and PA would simplify production and confer a broader spectrum of protection than that seen with PA alone.

Pharmacokinetics and efficacy of a vaginally administered maraviroc gel in rhesus macaques

Objectives: To investigate the pharmacokinetics (PK) of maraviroc, a CCR5-targeted HIV-1 entry inhibitor, in rhesus macaques following vaginal administration of various maraviroc-loaded aqueous hydroxyethylcellulose (HEC) gels, and to correlate the PK data with efficacy in a single high-dose vaginal SHIV-162P3 challenge model.

Methods: Maraviroc concentrations in vaginal fluid (Weck-Cel® sponge), vaginal tissue (punch biopsy) and plasma were assessed over 72 h following single dose vaginal application of various maraviroc-loaded HEC gels. The range of maraviroc gel concentrations was sufficiently broad (0.003 – 3.3% w/w) such that test gels included both fully solubilised and predominantly dispersed formulations. The efficacy of the HEC gels against a single high dose vaginal SHIV-162P3 challenge was also measured, and correlated with the PK concentrations.

Results: Maraviroc concentrations in vaginal fluid (range 104 – 107 ng/mL), vaginal tissue (100-1200 ng/g) and plasma (< 102 ng/mL) were highly dependent on maraviroc gel loading, irrespective of the form of the maraviroc component within the gel (solubilised vs. dispersed). Fluid and plasma concentrations were generally highest 0.5 or 2 h after gel application, before declining steadily out to 72 h. Maraviroc concentrations in the various biological compartments correlated strongly with the extent of protection against vaginal SHIV-162P3 challenge. Complete protection was achieved with a 3.3% w/w maraviroc gel.

Conclusions: A high degree of correlation between PK and efficacy was observed. Based on the data obtained with the 3.3% w/w maraviroc gel, maintenance of vaginal fluid and tissue levels in the order of 107 ng/mL and 103 ng/g, respectively, are required for complete protection with this compound.

Survival and persistence of opportunistic Burkholderia species in host cells

Burkholderia are microorganisms that have a unique ability to adapt and survive in many different environments. They can also serve as biopesticides and be used for the biodegradation of organic compounds. Usually harmless while living in the soil, these bacteria are opportunistic pathogens of plants and immunocompromised patients, and occasionally infect healthy individuals. Some of the species in this genus can also be utilised as biological weapons. They all possess very large genomes and have two or more circular chromosomes. Their survival and persistence, not only in the environment but also in host cells, offers a remarkable example of bacterial adaptation.

RpoS and RpoN are involved in the growth-dependent regulation of rfaH transcription and O antigen expression in Salmonella enterica serovar Typhi

We reported earlier that the production of O antigen lipopolysaccharide (LPS) by Salmonella enterica serovar Typhi (Salmonella typhi) increases at the onset of stationary phase and correlates with a growth-regulated expression of the rfaH gene under the control of the alternative sigma factor RpoN (Microbiology 148 (2002) 3789). In this study, we demonstrate that RpoS also modulates rfaH promoter activity as revealed by the absence of growth-dependent regulation of an rfaH-lacZ transcriptional fusion and O antigen production in a S. typhi rpoS mutant. Introduction of a constitutively expressed rpoN gene into the rpoS mutant restored increased production of O antigen during stationary phase, suggesting that constitutive production of RpoN could overcome the RpoS defect. Similar results were observed when an rpoS rpoN double mutant was transformed with the intact rpoN gene. Thus, we conclude that both RpoS and RpoN control the rfaH promoter activity and concomitantly, the production of O-specific LPS in S. typhi.

Identification of a general secretory pathway in a human isolate of Burkholderia vietnamiensis (formerly B. cepacia complex genomovar V) that is required for the secretion of hemolysin and phospholipase C activities

Members of the Burkholderia cepacia complex can secrete proteases, lipases, and hemolysins. We report in this study the identification of a general secretory pathway present in a B. vietnamiensis (formerly genomovar V) clinical isolate, which is required for the efficient secretion of phospholipase C and hemolysin activities. Southern blot hybridization experiments revealed that this general secretion pathway is highly conserved among the different genomovars of the B. cepacia complex and is homologous to a similar system described in B. pseudomallei. We also show that this pathway appears not to be necessary for intracellular survival of B. vietnamiensis within Acanthamoeba polyphaga.

Characterization of RarA, a Novel AraC Family Multidrug Resistance Regulator in Klebsiella pneumoniae

Transcriptional regulators, such as SoxS, RamA, MarA, and Rob, which upregulate the AcrAB efflux pump, have been shown to be associated with multidrug resistance in clinically relevant Gram-negative bacteria. In addition to the multidrug resistance phenotype, these regulators have also been shown to play a role in the cellular metabolism and possibly the virulence potential of microbial cells. As such, the increased expression of these proteins is likely to cause pleiotropic phenotypes. Klebsiella pneumoniae is a major nosocomial pathogen which can express the SoxS, MarA, Rob, and RamA proteins, and the accompanying paper shows that the increased transcription of ramA is associated with tigecycline resistance (M. Veleba and T. Schneiders, Antimicrob. Agents Chemother. 56:4466-4467, 2012). Bioinformatic analyses of the available Klebsiella genome sequences show that an additional AraC-type regulator is encoded chromosomally. In this work, we characterize this novel AraC-type regulator, hereby called RarA (Regulator of antibiotic resistance A), which is encoded in K. pneumoniae, Enterobacter sp. 638, Serratia proteamaculans 568, and Enterobacter cloacae. We show that the overexpression of rarA results in a multidrug resistance phenotype which requires a functional AcrAB efflux pump but is independent of the other AraC regulators. Quantitative real-time PCR experiments show that rarA (MGH 78578 KPN_02968) and its neighboring efflux pump operon oqxAB (KPN_02969_02970) are consistently upregulated in clinical isolates collected from various geographical locations (Chile, Turkey, and Germany). Our results suggest that rarA overexpression upregulates the oqxAB efflux pump. Additionally, it appears that oqxR, encoding a GntR-type regulator adjacent to the oqxAB operon, is able to downregulate the expression of the oqxAB efflux pump, where OqxR complementation resulted in reductions to olaquindox MICs.

Tigecycline Resistance Can Occur Independently of the ramA Gene in Klebsiella pneumoniae

Tigecycline resistance in Klebsiella pneumoniae results from ramA upregulation that causes the overexpression of the efflux pump, AcrAB-TolC. Tigecycline mutants, derived from Ecl8?ramA, can exhibit a multidrug resistance phenotype due to increased transcription of the marA, rarA, acrAB, and oqxAB genes. These findings support the idea that tigecycline or multidrug resistance in K. pneumoniae, first, is not solely dependent on the ramA gene, and second, can arise via alternative regulatory pathways in K. pneumoniae. © 2012, American Society for Microbiology.

FhCaBP4: A Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains

In trematodes, there is a family of proteins which combine EF-hand-containing domains with dynein light chain (DLC)-like domains. A member of this family from the liver fluke, Fasciola hepatica-FhCaBP4-has been identified and characterised biochemically. FhCaBP4 has an N-terminal domain containing two imperfect EF-hand sequences and a C-terminal dynein light chain-like domain. Molecular modelling predicted that the two domains are joined by a flexible linker. Native gel electrophoresis demonstrated that FhCaBP4 binds to calcium, manganese, barium and strontium ions, but not to magnesium or zinc ions. The hydrophobic, fluorescent probe 8-anilinonaphthalene-1-sulphonate bound more tightly to FhCaBP4 in the presence of calcium ions. This suggests that the protein undergoes a conformational change on ion binding which increases the number of non-polar residues on the surface. FhCaBP4 was protected from limited proteolysis by the calmodulin antagonist W7, but not by trifluoperazine or praziquantel. Protein-protein cross-linking experiments showed that FhCaBP4 underwent calcium ion-dependent dimerisation. Since DLCs are commonly dimeric, it is likely that FhCaBP4 dimerises through this domain. The molecular model reveals that the calcium ion-binding site is located close to a key sequence in the DLC-like domain, suggesting a plausible mechanism for calcium-dependent dimerisation.