- Volume 156, Issue 3, 2010

Bacillus subtilis is the model organism for a large group of Gram-positive bacteria, the Firmicutes. Several online databases have been established over time to manage its genetic and metabolic information, but they differ greatly in their rate of update and their focus on B. subtilis. Therefore, a European systems biology consortium called for an integrated solution that empowers its users to enrich online content. To meet this goal we created SubtiWiki and SubtiPathways, two complementary online tools for gene and pathway information on B. subtilis 168. SubtiWiki (http://subtiwiki.uni-goettingen.de/) is a scientific wiki for all genes of B. subtilis and their protein or RNA products. Each gene page contains a summary of the most important information; sections on the gene, its product and expression; sections concerning biological materials and laboratories; and a list of references. SubtiWiki has been seeded with key content and can be extended by any researcher after a simple registration, thus keeping it always up to date. As a complement, SubtiPathways (http://subtipathways.uni-goettingen.de/) is an online tool for navigation of the metabolism of B. subtilis and its regulation. Each SubtiPathways diagram presents a metabolic pathway with its participating enzymes, together with the regulatory mechanisms that act on their expression and activity, in an intuitive interface that is based on Google Maps. Together, SubtiWiki and SubtiPathways provide an integrated view of the processes that make up B. subtilis and its components, making it the most comprehensive web resource for B. subtilis researchers.

Rifampicin and its derivatives are at the forefront of the current standard chemotherapeutic regimen for active tuberculosis; they act by inhibiting the transcription activity of prokaryotic RNA polymerase. Rifampicin is believed to interact with the β subunit of RNA polymerase. However, it has been observed that protein–protein interactions with RNA polymerase core enzyme lead to its reduced susceptibility to rifampicin. This mechanism became more diversified with the discovery of RbpA, a novel RNA polymerase-binding protein, in Streptomyces coelicolor that could mitigate the effect of rifampicin on RNA polymerase activity. MsRbpA is a homologue of RbpA in Mycobacterium smegmatis. On deciphering the role of MsRbpA in M. smegmatis we found that it interacts with RNA polymerase and increases the rifampicin tolerance levels, both in vitro and in vivo. It interacts with the β subunit of RNA polymerase. However, it was found to be incapable of rescuing rifampicin-resistant RNA polymerases in the presence of rifampicin at the respective IC50.

Although dermatophytes are the most common agents of superficial mycoses in humans and animals, the molecular basis of the pathogenicity of these fungi is largely unknown. In vitro digestion of keratin by dermatophytes is associated with the secretion of multiple proteases, which are assumed to be responsible for their particular specialization to colonize and degrade keratinized host structures during infection. To investigate the role of individual secreted proteases in dermatophytosis, a guinea pig infection model was established for the zoophilic dermatophyte Arthroderma benhamiae, which causes highly inflammatory cutaneous infections in humans and rodents. By use of a cDNA microarray covering approximately 20–25 % of the A. benhamiae genome and containing sequences of at least 23 protease genes, we revealed a distinct in vivo protease gene expression profile in the fungal cells, which was surprisingly different from the pattern elicited during in vitro growth on keratin. Instead of the major in vitro-expressed proteases, others were activated specifically during infection. These enzymes are therefore suggested to fulfil important functions that are not exclusively associated with the degradation of keratin. Most notably, the gene encoding the serine protease subtilisin 6, which is a known major allergen in the related dermatophyte Trichophyton rubrum and putatively linked to host inflammation, was found to be the most strongly upregulated gene during infection. In addition, our approach identified other candidate pathogenicity-related factors in A. benhamiae, such as genes encoding key enzymes of the glyoxylate cycle and an opsin-related protein. Our work provides what we believe to be the first broad-scale gene expression profile in human pathogenic dermatophytes during infection, and points to putative virulence-associated mechanisms that make these micro-organisms the most successful aetiological agents of superficial mycoses.

The Burkholderia cepacia complex (Bcc) emerged as problematic opportunistic pathogens to cystic fibrosis (CF) patients. Although several virulence factors have been identified in Bcc, the knowledge of their relative contribution to Bcc pathogenicity remains scarce. In this work, we describe the identification and characterization of a B. cepacia IST408 mutant containing a disruption in the hfq gene. In other bacteria, Hfq is a global regulator of metabolism, acting as an RNA chaperone involved in the riboregulation of target mRNAs by small regulatory non-coding RNAs (sRNAs). The B. cepacia Hfq protein was overproduced as a histidine-tagged derivative, and we show evidence that the protein forms hexamers and binds sRNAs. When provided in trans, the B. cepacia IST408 hfq gene complemented the Escherichia coli hfq mutant strain GS081. Our results also show that the B. cepacia hfq mutant is more susceptible to stress conditions mimicking those faced by Bcc bacteria when infecting the CF host. In addition, the B. cepacia hfq mutant and two hfq mutants derived from B. dolosa and B. ambifaria clinical isolates also exhibited a reduced ability to colonize and kill the nematode Caenorhabditis elegans, used as an infection model. These data, together with the conservation of Hfq orthologues among Bcc, strongly suggest that Hfq plays a major role in the survival of Bcc under stress conditions, contributing to the success of Bcc as CF pathogens.

Device-associated microbial growth, including Candida biofilms, represents more than half of all human microbial infections and, despite a relatively small risk of implant-associated diseases, this type of infection usually leads to high morbidity, increased health-care costs and prolonged antimicrobial therapy. Animal models are needed to elucidate the complex host–pathogen interactions that occur during the development of attached and structured biofilm populations. We describe here a new in vivo model to study Candida biofilm, based on the avascular implantation of small catheters in rats. Polyurethane biomaterials challenged with Candida cells were placed underneath the skin of immunosuppressed animals following only minor surgery. The model allowed the study of up to ten biofilms at once, and the recovery of mature biofilms from 2 days after implantation. The adhering inoculum was adjusted to the standard threshold of positive diagnosis of fungal infection in materials recovered from patients. Wild-type biofilms were mainly formed of hyphal cells, and they were unevenly distributed across the catheter length as observed in infected materials in clinical cases. The hyphal multilayered structure of the biofilms of wild-type strains was observed by confocal microscopy and compared to the monolayer of yeast or hyphal cells of two well-known biofilm-deficient strains, efg1Δ/efg1Δ cph1Δ/cph1Δ and bcr1Δ/bcr1Δ, respectively. The subcutaneous Candida biofilm model relies on the use of implanted catheters with accessible, fast and minor surgery to the animals. This model can be used to characterize the ability of antimicrobial agents to eliminate biofilms, and to evaluate the prophylactic effect of antifungal drugs and biomaterial coatings.

The interaction of bacteria with platelets is implicated in the pathogenesis of endovascular infections, including infective endocarditis, of which Staphylococcus aureus is the leading cause. Several S. aureus surface proteins mediate aggregation of platelets by fibrinogen- or fibronectin-dependent processes, which also requires specific antibodies. In this study S. aureus was grown in iron-limited medium to mimic in vivo conditions in which iron is unavailable to pathogens. Under such conditions, a S. aureus mutant lacking the known platelet-activating surface proteins adhered directly to platelets in the absence of plasma proteins and triggered aggregation. Platelet adhesion and aggregation was prevented by inhibiting expression of iron-regulated surface determinant (Isd) proteins. Mutants defective in IsdB, but not IsdA or IsdH, were unable to adhere to or aggregate platelets. Antibodies to the platelet integrin GPIIb/IIIa inhibited platelet adhesion by IsdB-expressing strains, as did antagonists of GPIIb/IIIa. Surface plasmon resonance demonstrated that recombinant IsdB interacts directly with GPIIb/IIIa.

The isoleucine and valine biosynthetic enzyme acetolactate synthase (Ilv2p) is an attractive antifungal drug target, since the isoleucine and valine biosynthetic pathway is not present in mammals, Saccharomyces cerevisiae ilv2Δ mutants do not survive in vivo, Cryptococcus neoformans ilv2 mutants are avirulent, and both S. cerevisiae and Cr. neoformans ilv2 mutants die upon isoleucine and valine starvation. To further explore the potential of Ilv2p as an antifungal drug target, we disrupted Candida albicans ILV2, and demonstrated that Ca. albicans ilv2Δ mutants were significantly attenuated in virulence, and were also profoundly starvation-cidal, with a greater than 100-fold reduction in viability after only 4 h of isoleucine and valine starvation. As fungicidal starvation would be advantageous for drug design, we explored the basis of the starvation-cidal phenotype in both S. cerevisiae and Ca. albicans ilv2Δ mutants. Since the mutation of ILV1, required for the first step of isoleucine biosynthesis, did not suppress the ilv2Δ starvation-cidal defects in either species, the cidal phenotype was not due to α-ketobutyrate accumulation. We found that starvation for isoleucine alone was more deleterious in Ca. albicans than in S. cerevisiae, and starvation for valine was more deleterious than for isoleucine in both species. Interestingly, while the target of rapamycin (TOR) pathway inhibitor rapamycin further reduced S. cerevisiae ilv2Δ starvation viability, it increased Ca. albicans ilv1Δ and ilv2Δ viability. Furthermore, the recovery from starvation was dependent on the carbon source present during recovery for S. cerevisiae ilv2Δ mutants, reminiscent of isoleucine and valine starvation inducing a viable but non-culturable-like state in this species, while Ca. albicans ilv1Δ and ilv2Δ viability was influenced by the carbon source present during starvation, supporting a role for glucose wasting in the Ca. albicans cidal phenotype.

Phosphoribosylamine (PRA) is the first intermediate in the common purine/thiamine biosynthetic pathway and is primarily synthesized by the product of the purF gene, glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (E.C. 2.4.2.14). Past genetic and biochemical studies have shown that multiple mechanisms for the synthesis of PRA independent of PurF are present in Salmonella enterica. Here, we describe mutant alleles of the essential prsA gene, which encodes PRPP synthetase (E.C.2.7.6.1), that allow PurF-independent thiamine synthesis. The mutant alleles resulted in reduced PrsA activity in extracts, caused nutritional requirements indicative of PRPP limitation and allowed non-enzymic formation of PRA due to a build-up of ribose 5-phosphate (R5P). These results emphasize the balance that must be reached between pathways competing for the same substrate to maintain robustness of the metabolic network.

This study shows that the superoxide radical (O2 •−), a direct indicator of oxidative stress, is involved in the differentiation of the phytopathogenic filamentous fungi Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotium rolfsii and Sclerotinia minor, shown by using superoxide dismutase (SOD) mimetics to decrease their sclerotial differentiation. The production rate of O2 •− and SOD levels in these fungi, as expected, were significantly lowered by the SOD mimetics, with concomitant decrease of the indirect indicator of oxidative stress, lipid peroxidation.

Chromohalobacter salexigens DSM 3043, whose genome has been sequenced, is known to degrade (R,S)-sulfolactate as a sole carbon and energy source for growth. Utilization of the compound(s) was shown to be quantitative, and an eight-gene cluster (Csal_1764–Csal_1771) was hypothesized to encode the enzymes in the degradative pathway. It comprised a transcriptional regulator (SuyR), a Tripartite Tricarboxylate Transporter-family uptake system for sulfolactate (SlcHFG), two sulfolactate dehydrogenases of opposite sulfonate stereochemistry, namely novel SlcC and ComC [(R)-sulfolactate dehydrogenase] [EC1.1.1.272] and desulfonative sulfolactate sulfo-lyase (SuyAB) [EC4.4.1.24]. Inducible reduction of 3-sulfopyruvate, inducible SuyAB activity and induction of an unknown protein were detected. Separation of the soluble proteins from induced cells on an anion-exchange column yielded four relevant fractions. Two different fractions reduced sulfopyruvate with NAD(P)H, a third yielded SuyAB activity, and the fourth contained the unknown protein. The latter was identified by peptide-mass fingerprinting as SlcH, the candidate periplasmic binding protein of the transport system. Separated SuyB was also identified by peptide-mass fingerprinting. ComC was partially purified and identified by peptide-mass fingerprinting. The (R)-sulfolactate that ComC produced from sulfopyruvate was a substrate for SuyAB, which showed that SuyAB is (R)-sulfolactate sulfo-lyase. SlcC was purified to homogeneity. This enzyme also formed sulfolactate from sulfopyruvate, but the latter enantiomer was not a substrate for SuyAB. SlcC was obviously (S)-sulfolactate dehydrogenase.