Effect of stress on viral-bacterial synergy in bovine respiratory disease: novel mechanisms to regulate inflammation.

The severity of bovine respiratory infections has been linked to a variety of factors, including environmental and nutritional changes, transportation, and social reorganization of weaned calves. Fatal respiratory infections, however, usually occur when a primary viral infection compromises host defences and enhances the severity of a secondary bacterial infection. This viral-bacterial synergy can occur by a number of different mechanisms and disease challenge models have been developed to analyse host responses during these respiratory infections. A primary bovine herpesvirus-1 (BHV-1) respiratory infection followed by a secondary challenge with Mannheimia haemolytica results in fatal bovine respiratory disease (BRD) and host responses to these two pathogens have been studied extensively. We used this disease model to demonstrate that stress significantly altered the viral-bacterial synergy resulting in fatal BRD. Functional genomic analysis revealed that BHV-1 infection enhanced toll-like receptors (TLR) expression and increased pro-inflammatory responses which contribute to the severity of a Mannheimia haemolytica infection. TLRs play a critical role in detecting bacterial infections and inducing pro-inflammatory responses. It is difficult to understand, however, how stress-induced corticosteroids could enhance this form of viral-bacterial synergy. Nuclear translocation of the glucocorticoid receptor activates cell signalling pathways which inhibit both TLR signalling and pro-inflammatory responses. The apparent conundrum between stress-induced corticosteroids and enhanced BRD susceptibility is discussed in terms of present data and previous investigations of stress and respiratory disease.

PSORTb v.2.0: expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis.

MOTIVATION: PSORTb v.1.1 is the most precise bacterial localization prediction tool available. However, the program's predictive coverage and recall are low and the method is only applicable to Gram-negative bacteria. The goals of the present work are as follows: increase PSORTb's coverage while maintaining the existing precision level, expand it to include Gram-positive bacteria and then carry out a comparative analysis of localization. RESULTS: An expanded database of proteins of known localization and new modules using frequent subsequence-based support vector machines was introduced into PSORTb v.2.0. The program attains a precision of 96% for Gram-positive and Gram-negative bacteria and predictive coverage comparable to other tools for whole proteome analysis. We show that the proportion of proteins at each localization is remarkably consistent across species, even in species with varying proteome size. AVAILABILITY: Web-based version: http://www.psort.org/psortb. Standalone version: Available through the website under GNU General Public License. CONTACT: psort-mail@sfu.ca, brinkman@sfu.ca SUPPLEMENTARY INFORMATION: http://www.psort.org/psortb/supplementaryinfo.html.

Evolutionary relationships among virulence-associated histidine kinases.

A strong relationship between virulence-associated sensor histidine kinases of fungi and those in Streptomyces coelicolor was observed, and phylogenetic analysis suggested that bacterium-to-eukaryote horizontal gene transfer had occurred between ancestors of these organisms. Phylogenetic analysis also identified a group of histidine kinases orthologous to the Streptomyces proteins that includes Pseudomonas aeruginosa GacS. We provide evidence that GacS is important for swarming motility, lipase production, and virulence in mice and had evolved to have partial functional overlaps with PhoQ, a less-related virulence-associated histidine kinase.

PhyloBLAST: facilitating phylogenetic analysis of BLAST results.

PhyloBLAST is an internet-accessed application based on CGI/Perl programming that compares a users protein sequence to a SwissProt/TREMBL database using BLAST2 and then allows phylogenetic analyses to be performed on selected sequences from the BLAST output. Flexible features such as ability to input your own multiple sequence alignment and use PHYLIP program options provide additional web-based phylogenetic analysis functionality beyond the analysis of a BLAST result.

Evaluation of a structural model of Pseudomonas aeruginosa outer membrane protein OprM, an efflux component involved in intrinsic antibiotic resistance.

The outer membrane protein OprM of Pseudomonas aeruginosa is involved in intrinsic and mutational multiple-antibiotic resistance as part of two resistance-nodulation-division efflux systems. The crystal structure of TolC, a homologous protein in Escherichia coli, was recently published (V. Koronakis, A. Sharff, E. Koronakis, B. Luisl, and C. Hughes, Nature 405:914-919, 2000), demonstrating a distinctive architecture comprising outer membrane beta-barrel and periplasmic helical-barrel structures, which assemble differently from the common beta-barrel-only conformation of porins. Based on their sequence similarity, a similar content of alpha-helical and beta-sheet structure determined by circular dichroism spectroscopy, and our observation that OprM, like TolC, reconstitutes channels in planar bilayer membranes, OprM and TolC were considered to be structurally homologous, and a model of OprM was constructed by threading its sequence to the TolC crystal structure. Residues thought to be important for the TolC structure were conserved in space in this OprM model. Analyses of deletion mutants and previously isolated insertion mutants of OprM in the context of this model allowed us to propose roles for different protein domains. Our data indicate that the helical barrel of the protein is critical for both the function and the integrity of the protein, while a C-terminal domain localized around the equatorial plane of this helical barrel is dispensable. Extracellular loops appear to play a lesser role in substrate specificity for this efflux protein compared to classical porins, and there appears to be a correlation between the change in antimicrobial activity for OprM mutants and the pore size. Our model and channel formation studies support the "iris" mechanism of action for TolC and permit us now to form more focused hypotheses about the functional domains of OprM and its related family of efflux proteins.

Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the top three causes of opportunistic human infections. A major factor in its prominence as a pathogen is its intrinsic resistance to antibiotics and disinfectants. Here we report the complete sequence of P. aeruginosa strain PAO1. At 6.3 million base pairs, this is the largest bacterial genome sequenced, and the sequence provides insights into the basis of the versatility and intrinsic drug resistance of P. aeruginosa. Consistent with its larger genome size and environmental adaptability, P. aeruginosa contains the highest proportion of regulatory genes observed for a bacterial genome and a large number of genes involved in the catabolism, transport and efflux of organic compounds as well as four potential chemotaxis systems. We propose that the size and complexity of the P. aeruginosa genome reflect an evolutionary adaptation permitting it to thrive in diverse environments and resist the effects of a variety of antimicrobial substances.

The amino terminus of Pseudomonas aeruginosa outer membrane protein OprF forms channels in lipid bilayer membranes: correlation with a three-dimensional model.

Pseudomonas aeruginosa OprF forms 0.36-nS channels and, rarely, 2- to 5-nS channels in lipid bilayer membranes. We show that a protein comprising only the N-terminal 162-amino-acid domain of OprF formed the smaller, but not the larger, channels in lipid bilayers. Circular dichroism spectroscopy indicated that this protein folds into a beta-sheet-rich structure, and three-dimensional comparative modeling revealed that it shares significant structural similarity with the amino terminus of the orthologous protein Escherichia coli OmpA, which has been shown to form a beta-barrel. OprF and OmpA share only 15% identity in this domain, yet these results support the utility of modeling such widely divergent beta-barrel domains in three dimensions in order to reveal similarities not readily apparent through primary sequence comparisons. The model is used to further hypothesize why porin activity differs for the N-terminal domains of OprF and OmpA.

Influence of a putative ECF sigma factor on expression of the major outer membrane protein, OprF, in Pseudomonas aeruginosa and Pseudomonas fluorescens.

The gene encoding OprF, a major outer membrane protein in Pseudomonas species (formerly known as type 1 pseudomonads), was thought to be constitutively transcribed from a single sigma 70 promoter immediately upstream of the gene. We now report the identification of a novel putative ECF (extracytoplasmic function) sigma factor gene, sigX, located immediately upstream of oprF in both Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens OE 28.3 and show that disruption of this gene significantly reduces OprF expression. In P. aeruginosa, Northern analysis demonstrated that this reduction was a result of an effect on transcription of monocistronic oprF combined with a polar effect due to termination of a transcript containing sigX and oprF. Comparison of sigX-disrupted and wild-type cell transcripts by primer extension indicated that monocistronic transcription of oprF occurs from two overlapping promoters, one that is SigX-dependent and resembles ECF sigma factor promoters in its minus-35 region and another promoter that is independent of SigX and is analogous to the sigma 70-type promoter previously reported. Complementation of the P. aeruginosa sigX-disrupted mutant with plasmid-encoded OprF did not resolve the phenotypes associated with this mutant, which included a markedly reduced logarithmic-phase growth rate in rich medium (compared to that in minimal medium), further reduction of the growth rate in a low-osmolarity environment, secretion of an unidentified pigment, and increased sensitivity to the antibiotic imipenem. This indicates that SigX is involved in the regulation of other genes in P. aeruginosa. Disruption of the sigX gene in P. fluorescens also had an effect on the logarithmic-phase growth rate in rich medium. A conserved sigX gene was also identified in a Pseudomonas syringae isolate and six P. aeruginosa clinical isolates. Collectively, these data indicate that an ECF sigma factor plays a role in the regulation and expression of OprF and also affects other genes.

Roles of the carboxy-terminal half of Pseudomonas aeruginosa major outer membrane protein OprF in cell shape, growth in low-osmolarity medium, and peptidoglycan association.

OprF, the major outer membrane protein of Pseudomonas aeruginosa, is multifunctional in that it can act as a nonspecific porin, plays a role in the maintenance of cell shape, and is required for growth in a low-osmolarity environment. The latter two structural roles of OprF, and OprF's association with the peptidoglycan, have been proposed to be localized in the carboxy terminus of the protein, based on this region's similarity to members of the OmpA family of proteins. To determine if this is correct, we constructed a series of C-terminally truncated OprF derivatives and examined their effects on P. aeruginosa cell length and growth in low-osmolarity medium. While the C terminus of OprF was required for wild-type cell length and growth in low-osmolarity medium, expression of the N terminus (first 163 amino acids [aa]) also influenced these phenotypes (compared with OprF deficiency). The first 154 to 164 aa of OprF seemed required for stable protein expression, consistent with the existence of a beta-barrel domain in the N terminus of OprF. Greater than 215 aa of the protein were required for strong peptidoglycan association, confirming that residues in the C-terminal end of OprF are required for peptidoglycan binding. OprF deficiency did not affect the in vivo growth of an OprF-deficient strain in a mouse chamber model. Collectively, these data suggest that the C terminus of OprF plays a role in cell length, growth of P. aeruginosa in low-osmolarity media (but not in vivo), and peptidoglycan association, while the N terminus has an influence on the first two characteristics and is additionally important for stable protein expression.