Dietary trehalose enhances virulence of epidemic Clostridium difficile.

Clostridium difficile disease has recently increased to become a dominant nosocomial pathogen in North America and Europe, although little is known about what has driven this emergence. Here we show that two epidemic ribotypes (RT027 and RT078) have acquired unique mechanisms to metabolize low concentrations of the disaccharide trehalose. RT027 strains contain a single point mutation in the trehalose repressor that increases the sensitivity of this ribotype to trehalose by more than 500-fold. Furthermore, dietary trehalose increases the virulence of a RT027 strain in a mouse model of infection. RT078 strains acquired a cluster of four genes involved in trehalose metabolism, including a PTS permease that is both necessary and sufficient for growth on low concentrations of trehalose. We propose that the implementation of trehalose as a food additive into the human diet, shortly before the emergence of these two epidemic lineages, helped select for their emergence and contributed to hypervirulence.

Direct detection of extended-spectrum beta-lactamases (CTX-M) from blood cultures by LC-MS/MS bottom-up proteomics.

Rapid bacterial species identification and antibiotic susceptibility testing in positive blood cultures have an important impact on the antibiotic treatment for patients. To identify extended-spectrum beta-lactamases (ESBL) directly in positive blood culture bottles, we developed a workflow of saponin extraction followed by a bottom-up proteomics approach using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The workflow was applied to positive blood cultures with Escherichia coli and Klebsiella pneumoniae collected prospectively in two academic hospitals over a 4-month period. Of 170 positive blood cultures, 22 (12.9%) contained ESBL-positive isolates based on standard susceptibility testing. Proteomic analysis identified CTX-M ESBLs in 95% of these isolates directly in positive blood cultures, whereas no false positives were found in the non-ESBL producing positive blood cultures. The results were confirmed by molecular characterisation of beta-lactamase genes. Based on this proof-of-concept study, we conclude that LC-MS/MS-based protein analysis can directly identify extended-spectrum beta lactamases in E. coli and K. pneumoniae positive blood cultures, and could be further developed for application in routine diagnostics.

Retargeting of the mitochondrial protein p32/gC1Qr to a cytoplasmic compartment and the cell surface.

p32/gC1qR is a small acidic protein that has been reported to have a broad range of distinct functions and to associate with a wide array of cellular, viral and bacterial proteins. It has been found in each of the main cellular compartments including mitochondria, nucleus and cytoplasm and is also thought to be located at the plasma membrane and secreted into the extracellular matrix. The true physiological role(s) of p32 remains controversial because it has been difficult to reconcile all of the findings on protein interactions and the seemingly disparate observations on compartmentalisation. However, it has been proposed that p32 is somehow involved in transport processes connecting diverse cellular compartments and the cell surface. Here we show that native p32 appears to be localised mainly in the mitochondria and is not detectable on the cell surface. However, addition of a short tag to the N-terminus of p32 appears to block its mitochondrial targeting, resulting in redirection into a cytoplasmic vesicular pattern, overlapping with the endoplasmic reticulum. The redirection of p32 results in an alteration in and co-localisation with ER markers including calreticulin, a lumenal ER chaperone. Furthermore, we show both by immunofluorescence and cross-linking studies that this also results in cell-surface expression of p32. These results indicate that, at least under certain circumstances, p32 can be retargeted and may help to provide an explanation for the diverse observations on its localization.

The multifunctional regulatory proteins ABF1 and CPF1 are involved in the formation of a nuclease-hypersensitive region in the promoter of the QCR8 gene.

The abundant DNA-binding proteins ABF1 and CPF1 are members of a family of global regulators with diverse chromosomal functions in the yeast Saccharomyces cerevisiae. Recent evidence suggests that these protein factors may be involved in establishing and maintaining well-defined chromatin in promoter regions and other genetic elements. We have investigated the involvement of ABF1 and CPF1 in chromatin organization at the QCR8 gene, encoding subunit VIII of the mitochondrial ubiquinol-cytochrome c oxidoreductase. The promoter region of the QCR8 gene contains overlapping binding sites for ABF1 and CPF1. Nucleosome positioning studies indicate that the QCR8 gene is associated with a phased array of nucleosomes under both catabolite-repressed and derepressed growth conditions. Analysis of binding site mutants reveals that both ABF1 and CPF1 are involved in maintaining a nuclease-hypersensitive region in the QCR8 promoter. The chromatin structure at QCR8 during steady-state growth is, however, mainly dependent on binding of ABF1 to the promoter region. Implications of these findings for the role played by ABF1 and CPF1 in the regulation of mitochondrial biogenesis and other processes important for cell growth and division will be discussed.

The Oct-1 POU homeodomain stabilizes the adenovirus preinitiation complex via a direct interaction with the priming protein and is displaced when the replication fork passes.

Initiation of adenovirus DNA replication is strongly enhanced by two cellular transcription factors, NFI and Oct-1, which bind to the auxiliary origin and tether the viral precursor terminal protein-DNA polymerase (pTP.pol) complex to the core origin. NFI acts through a direct contact with the DNA polymerase, but the mode of action of Oct 1 is unknown. Employing glutathione S-transferase-POU pull-down assays and protein affinity chromatography, we have established that the POU domain contacts pTP rather than pol. The POU homeodomain is responsible for this interaction. The protein-protein contacts lead to increased binding of pTP-pol to the core origin, which is caused by a reduced off-rate. The enhanced formation of a pTP.pol.POU complex on the origin correlates with stimulation of replication. Using an immobilized replication system, we have studied the kinetics of dissociation of the Oct-1 POU domain during replication. In contrast to NFI, which dissociates very early in initiation, Oct-1 dissociates only when the binding site is rendered single-stranded upon translocation of the replication fork. Our data indicate that NFI and Oct-1 enhance initiation synergistically by touching different targets in the preinitiation complex and dissociate independently after initiation.

Linker length and composition influence the flexibility of Oct-1 DNA binding.

POU domain transcription factors have two separate helix-turn-helix DNA-binding subdomains, the POU homeodomain (POUhd) and the POU-specific domain (POUs). Each subdomain recognizes a specific subsite of 4 or 5 bp in the octamer recognition sequence. The Oct-1 POU subdomains are connected by a 23 amino acid unstructured linker region. To investigate the requirements for the linker and its role in DNA recognition, we constructed POU domains in which the subdomains are connected with linkers varying in length between 2 and 37 amino acids. Binding to the natural octamer site required a minimal linker length of between 10 and 14 amino acids. A POU domain with an eight amino acid linker, however, had a high affinity for a site in which the POUs recognition sequence was inverted. Computer modelling shows that inversion of the POUs subdomain shortens the distance between the subdomains sufficiently to enable an eight amino acid linker to bridge the distance. DNase I footprinting as well as mutation of the POUs-binding site confirms the inverted orientation of the POUs domain. Switching of the POUs and POUhd subdomains and separation by 3 bp leads to a large distance which could only be bridged effectively by a long 37 amino acid linker. In addition to linker length, mutation of a conserved glutamate residue in the linker affected binding. As shown by surface plasmon resonance measurements, this was caused by a decrease in the on-rate. Our data indicate that there are both length and sequence requirements in the linker region which allow flexibility leading to selective binding to differently spaced and oriented subsites.

Mutation of the Oct-1 POU-specific recognition helix leads to altered DNA binding and influences enhancement of adenovirus DNA replication.

To assess which residues of Oct-1 POU-specific (POUs) are important for DNA recognition and stimulation of adenovirus DNA replication we have mutated 10 residues of the POUs helix-turn-helix motif implicated in DNA contact. Seven of these turned out to have reduced DNA binding affinity. Of these, three alanine substituted proteins were found to have a changed specificity using a binding site selection procedure. Mutation of the first residue in the recognition helix, Gln44, to alanine led to a loss of specificity for the first two bases, TA, of the wild-type recognition site TATGC(A/T)AAT. Instead of the A, a T was selected, suggesting a new contact and a novel specificity. A change in specificity was also observed for the T45A mutant, which could bind to TATAC(A/T)AAT, a site hardly recognized by the wild-type protein. Mutation of residue Arg49 led to a relaxed specificity for three consecutive bases, TGC. This residue, which is critical for high affinity binding, is absent from the structurally homologous lambdoid helix-turn-helix motifs. Employing a reconstituted system all but two mutants could stimulate adenovirus DNA replication upon saturation. Mutation of residues Gln27 and Arg49 impairs the ability of the Oct-1 POU domain protein to enhance replication, with a concomitant loss of DNA contacts. Since the POU domain binds the precursor terminal protein-DNA polymerase complex and guides it to the origin, lack of stimulation may be caused by incorrect targetting of the DNA polymerase due to loss of specificity.

The DNA binding specificity of the bipartite POU domain and its subdomains.

The POU domain is a conserved DNA binding region of approximately 160 amino acids present in a family of eukaryotic transcription factors that play regulatory roles in development. The POU domain consists of two subdomains, the POU-specific (POUS) domain and a POU-type homeodomain (POUHD). We show here that, like the POUHD, the Oct-1 POUS domain can bind autonomously to DNA but with low affinity. DNA binding studies and in vitro binding site selection revealed that the POU subdomains each have a different sequence specificity. The binding consensus of the POUS domain [gAATAT(G/T)CA] and POUHD (RTAATNA) respectively overlap the 'left half' and right half' of the POU domain recognition sequence [a(a/t)TATGC(A/T) AAT(t/a)t]. In addition to the core sequence, which is very similar to the octamer motif (ATGCAAAT), the flanking bases make a significant contribution to the binding affinity of the POU domain. Interestingly, at some positions the sequence preferences of the isolated POU subdomains are distinct from those of the POU domain, suggesting that the POU domain binding site is more than a simple juxtaposition of the POUS and POUHD target sequences. In addition, analysis of the binding kinetics of the POU domain and POUHD indicates that the POUS domain enhances the binding affinity by reducing the dissociation rate. Our results show that the POU domain proteins have DNA binding properties distinct from those of classic homeodomain proteins. We suggest a model for the way in which an additional conserved domain adds further specificity to DNA recognition by homeodomain proteins.

Solution structure of the Oct-1 POU homeodomain determined by NMR and restrained molecular dynamics.

The POU homeodomain (POUhd), a divergent member of the well-studied class of homeodomain proteins, is the C-terminal part of the bipartite POU domain, the conserved DNA-binding domain of the POU proteins. In this paper we present the solution structure of POUhd of the human Oct-1 transcription factor. This fragment was overexpressed in Escherichia coli and studied by two- and three-dimensional homo- and heteronuclear NMR techniques, resulting in virtually complete 1H and 15N resonance assignments for residues 2-60. Using distance and dihedral constraints derived from the NMR data, 50 distance geometry structures were calculated, which were refined by means of restrained molecular dynamics. From this set a total of 31 refined structures were selected, having low constraint energy and few constraint violations. The ensemble of 31 structures displays a root-mean-square deviation of the coordinates of 0.59 A with respect to the average structure, calculated over the backbone atoms of residues 6 to 54. The fold of POUhd is very similar to that of the canonical homeodomains. Interestingly, the recognition helix of the free POUhd ends at residue 53, while in the cocrystal structure of the intact POU domain with the DNA octamer motif [Klemm, J.D., Rould, M.A., Aurora, R., Herr, W. and Pabo, C.O. (1994) Cell, 77, 21-32] this helix in the POUhd subdomain is extended as far as residue 60.