Impact of quorum sensing on fitness of Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, cell-cell communication based on N-acyl-homoserine lactone (AHL) signal molecules (termed quorum sensing) is known to control the production of extracellular virulence factors. Hence, in pathogenic interactions with host organisms, the quorum-sensing (QS) machinery can confer a selective advantage on P. aeruginosa. However, as shown by transcriptomic and proteomic studies, many intracellular metabolic functions are also regulated by quorum sensing. Some of these serve to regenerate the AHL precursors methionine and S-adenosyl-methionine and to degrade adenosine via inosine and hypoxanthine. The fact that a significant percentage of clinical and environmental isolates of P. aeruginosa is defective for QS because of mutation in the major QS regulatory gene lasR, raises the question of whether the QS machinery can have a negative impact on the organism's fitness. In vitro, lasR mutants have a higher probability to escape lytic death in stationary phase under alkaline conditions than has the QS-proficient wild type. Similar selective forces might also operate in natural environments.

Novel role of a family of major facilitator transporters in biofilm development and virulence of Candida albicans.

The QDR (quinidine drug resistance) family of genes encodes transporters belonging to the MFS (major facilitator superfamily) of proteins. We show that QDR transporters, which are localized to the plasma membrane, do not play a role in drug transport. Hence, null mutants of QDR1, QDR2 and QDR3 display no alterations in susceptibility to azoles, polyenes, echinocandins, polyamines or quinolines, or to cell wall inhibitors and many other stresses. However, the deletion of QDR genes, individually or collectively, led to defects in biofilm architecture and thickness. Interestingly, QDR-lacking strains also displayed attenuated virulence, but the strongest effect was observed with qdr2∆, qdr3∆ and in qdr1/2/3∆ strains. Notably, the attenuated virulence and biofilm defects could be reversed upon reintegration of QDR genes. Transcripts profiling confirmed differential expression of many biofilm and virulence-related genes in the deletion strains as compared with wild-type Candida albicans cells. Furthermore, lipidomic analysis of QDR-deletion mutants suggests massive remodelling of lipids, which may affect cell signalling, leading to the defect in biofilm development and attenuation of virulence. In summary, the results of the present study show that QDR paralogues encoding MFS antiporters do not display conserved functional linkage as drug transporters and perform functions that significantly affect the virulence of C. albicans.

Bacterial subfamily of LuxR regulators that respond to plant compounds.

Pseudomonas fluorescens are rhizobacteria known for their biocontrol properties. Several antimicrobial functions are crucial for this process, and the experiments described here investigate the modulation of their expression during the plant-bacterium interaction. The role of a LuxR family regulator in interkingdom signaling has been investigated using genome-scale transcriptome analysis, gene promoter studies in vivo and in vitro, biocontrol assays, and response to plant compounds. PsoR, a LuxR solo or orphan regulator of P. fluorescens, was identified. PsoR is solubilized and activates a lux-box-containing promoter only in the presence of macerated plants, suggesting the presence of a plant molecule(s) that most likely binds to PsoR. Gene expression profiles revealed that genes involved in the inhibition of plant pathogens were affected by PsoR, including a chitinase gene, iron metabolism genes, and biosynthetic genes of antifungal compounds. 2,4-Diacetylphloroglucinol production is PsoR dependent both in vitro and in vivo. psoR mutants were significantly reduced for their ability to protect wheat plants from root rot, and damping-off caused by Pythium ultimum infection. PsoR most likely senses a molecule(s) in the plant and modulates expression of genes that have a role in biocontrol. PsoR and related proteins form a subfamily of LuxR family regulators in plant-associated bacteria.

Proliferation is a prerequisite for bacterial superantigen-induced T cell apoptosis in vivo.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that binds to major histocompatibility complex class II molecules and selectively interacts with T cells that bear certain T cell receptor (TCR) V beta domains. Administration of SEB in adult mice results in initial proliferation of V beta 8+ T cells followed by a state of unresponsiveness resulting from a combination of clonal deletion and clonal anergy in the SEB-reactive population. At this time, it is unclear what relationship exists between the T cells that have proliferated and those that have been deleted or have become anergic. Here we show that only a fraction of the potentially reactive V beta 8+ T cells proliferate in response to SEB in vivo, and that all the cells that have proliferated eventually undergo apoptosis. Virtually no apoptosis can be detected in the nonproliferating V beta 8+ T cells. These data demonstrate a causal relationship between proliferation and apoptosis in response to SEB in vivo, and they further indicate that T cells bearing the same TCR V beta segment can respond differently to the same superantigen. The implications of this differential responsiveness in terms of activation and tolerance are discussed.

Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin.

The antimicrobial metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) contributes to the capacity of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soilborne pathogens. A 2, 4-DAPG-negative Tn5 insertion mutant of strain CHA0 was isolated, and the nucleotide sequence of the 4-kb genomic DNA region adjacent to the Tn5 insertion site was determined. Four open reading frames were identified, two of which were homologous to phlA, the first gene of the 2,4-DAPG biosynthetic operon, and to the phlF gene encoding a pathway-specific transcriptional repressor. The Tn5 insertion was located in an open reading frame, tentatively named phlH, which is not related to known phl genes. In wild-type CHA0, 2, 4-DAPG production paralleled expression of a phlA'-'lacZ translational fusion, reaching a maximum in the late exponential growth phase. Thereafter, the compound appeared to be degraded to monoacetylphloroglucinol by the bacterium. 2,4-DAPG was identified as the active compound in extracts from culture supernatants of strain CHA0 specifically inducing phlA'-'lacZ expression about sixfold during exponential growth. Induction by exogenous 2,4-DAPG was most conspicuous in a phlA mutant, which was unable to produce 2, 4-DAPG. In a phlF mutant, 2,4-DAPG production was enhanced severalfold and phlA'-'lacZ was expressed at a level corresponding to that in the wild type with 2,4-DAPG added. The phlF mutant was insensitive to 2,4-DAPG addition. A transcriptional phlA-lacZ fusion was used to demonstrate that the repressor PhlF acts at the level of transcription. Expression of phlA'-'lacZ and 2,4-DAPG synthesis in strain CHA0 was strongly repressed by the bacterial extracellular metabolites salicylate and pyoluteorin as well as by fusaric acid, a toxin produced by the pythopathogenic fungus Fusarium. In the phlF mutant, these compounds did not affect phlA'-'lacZ expression and 2, 4-DAPG production. PhlF-mediated induction by 2,4-DAPG and repression by salicylate of phlA'-'lacZ expression was confirmed by using Escherichia coli as a heterologous host. In conclusion, our results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites. This mechanism, which depends on phlF function, may help P. fluorescens to produce homeostatically balanced amounts of extracellular metabolites.

Gain of function mutations in CgPDR1 of Candida glabrata not only mediate antifungal resistance but also enhance virulence.

CgPdr1p is a Candida glabrata Zn(2)-Cys(6) transcription factor involved in the regulation of the ABC-transporter genes CgCDR1, CgCDR2, and CgSNQ2, which are mediators of azole resistance. Single-point mutations in CgPDR1 are known to increase the expression of at least CgCDR1 and CgCDR2 and thus to contribute to azole resistance of clinical isolates. In this study, we investigated the incidence of CgPDR1 mutations in a large collection of clinical isolates and tested their relevance, not only to azole resistance in vitro and in vivo, but also to virulence. The comparison of CgPDR1 alleles from azole-susceptible and azole-resistant matched isolates enabled the identification of 57 amino acid substitutions, each positioned in distinct CgPDR1 alleles. These substitutions, which could be grouped into three different "hot spots," were gain of function (GOF) mutations since they conferred hyperactivity to CgPdr1p revealed by constitutive high expression of ABC-transporter genes. Interestingly, the major transporters involved in azole resistance (CgCDR1, CgCDR2, and CgSNQ2) were not always coordinately expressed in presence of specific CgPDR1 GOF mutations, thus suggesting that these are rather trans-acting elements (GOF in CgPDR1) than cis-acting elements (promoters) that lead to azole resistance by upregulating specific combinations of ABC-transporter genes. Moreover, C. glabrata isolates complemented with CgPDR1 hyperactive alleles were not only more virulent in mice than those with wild type alleles, but they also gained fitness in the same animal model. The presence of CgPDR1 hyperactive alleles also contributed to fluconazole treatment failure in the mouse model. In conclusion, this study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo azole resistance but that they can also confer a selective advantage under host conditions.

Production of Pseudomonas aeruginosa Intercellular Small Signaling Molecules in Human Burn Wounds

Pseudomonas aeruginosa has developed a complex cell-to-cell communication system that relies on low-molecular weight excreted molecules to control the production of its virulence factors. We previously characterized the transcriptional regulator MvfR, that controls a major network of acute virulence functions in P. aeruginosa through the control of its ligands, the 4-hydroxy-2-alkylquinolines (HAQs)-4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS). Though HHQ and PQS are produced in infected animals, their ratios differ from those in bacterial cultures. Because these molecules are critical for the potency of activation of acute virulence functions, here we investigated whether they are also produced during human P. aeruginosa acute wound infection and whether their ratio is similar to that observed in P. aeruginosa-infected mice. We found that a clinically relevant P. aeruginosa isolate produced detectable levels of HAQs with ratios of HHQ and PQS that were similar to those produced in burned and infected animals, and not resembling ratios in bacterial cultures. These molecules could be isolated from wound tissue as well as from drainage liquid. These results demonstrate for the first time that HAQs can be isolated and quantified from acute human wound infection sites and validate the relevance of previous studies conducted in mammalian models of infection.

Examining chemical compound biodegradation at low concentrations through bacterial cell proliferation.

We show proof of principle for assessing compound biodegradation at 1-2 mg C per L by measuring microbial community growth over time with direct cell counting by flow cytometry. The concept is based on the assumption that the microbial community will increase in cell number through incorporation of carbon from the added test compound into new cells in the absence of (as much as possible) other assimilable carbon. We show on pure cultures of the bacterium Pseudomonas azelaica that specific population growth can be measured with as low as 0.1 mg 2-hydroxybiphenyl per L, whereas in mixed community 1 mg 2-hydroxybiphenyl per L still supported growth. Growth was also detected with a set of fragrance compounds dosed at 1-2 mg C per L into diluted activated sludge and freshwater lake communities at starting densities of 10(4) cells per ml. Yield approximations from the observed community growth was to some extent in agreement with standard OECD biodegradation test results for all, except one of the examined compounds.

Bacterial contamination of platelet concentrates: pathogen detection and inactivation methods

Whereas the reduction of transfusion related viral transmission has been a priority during the last decade, bacterial infection transmitted by transfusion still remains associated to a high morbidity and mortality, and constitutes the most frequent infectious risk of transfusion. This problem especially concerns platelet concentrates because of their favorable bacterial growth conditions. This review gives an overview of platelet transfusion-related bacterial contamination as well as on the different strategies to reduce this problem by using either bacterial detection or inactivation methods.

Contribution of CgPDR1-Regulated Genes in Enhanced Virulence of Azole-Resistant Candida glabrata.

In Candida glabrata, the transcription factor CgPdr1 is involved in resistance to azole antifungals via upregulation of ATP binding cassette (ABC)-transporter genes including at least CgCDR1, CgCDR2 and CgSNQ2. A high diversity of GOF (gain-of-function) mutations in CgPDR1 exists for the upregulation of ABC-transporters. These mutations enhance C. glabrata virulence in animal models, thus indicating that CgPDR1 might regulate the expression of yet unidentified virulence factors. We hypothesized that CgPdr1-dependent virulence factor(s) should be commonly regulated by all GOF mutations in CgPDR1. As deduced from transcript profiling with microarrays, a high number of genes (up to 385) were differentially regulated by a selected number (7) of GOF mutations expressed in the same genetic background. Surprisingly, the transcriptional profiles resulting from expression of GOF mutations showed minimal overlap in co-regulated genes. Only two genes, CgCDR1 and PUP1 (for PDR1upregulated and encoding a mitochondrial protein), were commonly upregulated by all tested GOFs. While both genes mediated azole resistance, although to different extents, their deletions in an azole-resistant isolate led to a reduction of virulence and decreased tissue burden as compared to clinical parents. As expected from their role in C. glabrata virulence, the two genes were expressed as well in vitro and in vivo. The individual overexpression of these two genes in a CgPDR1-independent manner could partially restore phenotypes obtained in clinical isolates. These data therefore demonstrate that at least these two CgPDR1-dependent and -upregulated genes contribute to the enhanced virulence of C. glabrata that acquired azole resistance.

Mechanism, regulation, and ecological role of bacterial cyanide biosynthesis.

A few bacterial species are known to produce and excrete hydrogen cyanide (HCN), a potent inhibitor of cytochrome c oxidase and several other metalloenzymes. In the producer strains, HCN does not appear to have a role in primary metabolism and is generally considered a secondary metabolite. HCN synthase of proteobacteria (especially fluorescent pseudomonads) is a membrane-bound flavoenzyme that oxidizes glycine, producing HCN and CO2. The hcnABC structural genes of Pseudomonas fluorescens and P. aeruginosa have sequence similarities with genes encoding various amino acid dehydrogenases/oxidases, in particular with nopaline oxidase of Agrobacterium tumefaciens. Induction of the hcn genes of P. fluorescens by oxygen limitation requires the FNR-like transcriptional regulator ANR, an ANR recognition sequence in the -40 region of the hcn promoter, and nonlimiting amounts of iron. In addition, expression of the hcn genes depends on a regulatory cascade initiated by the GacS/GacA (global control) two-component system. This regulation, which is typical of secondary metabolism, manifests itself during the transition from exponential to stationary growth phase. Cyanide produced by P. fluorescens strain CHA0 has an ecological role in that this metabolite accounts for part of the biocontrol capacity of strain CHA0, which suppresses fungal diseases on plant roots. Cyanide can also be a ligand of hydrogenases in some anaerobic bacteria that have not been described as cyanogenic. However, in this case, as well as in other situations, the physiological function of cyanide is unknown.

Role of bacterial biomass in the sorption of Ni by biomass-birnessite assemblages

Birnessites precipitated by bacteria are typically poorly crystalline Mn(IV) oxides enmeshed within biofilms to form complex biomass-birnessite assemblages. The strong sorption affinity of bacteriogenic birnessites for environmentally important trace metals is relatively well understood mechanistically, but the role of bacterial cells and extracellular polymeric substances appears to vary among trace metals. To assess the role of biomass definitively, comparison between metal sorption by biomass at high metal loadings in the presence and absence of birnessite is required. We investigated the biomass effect on Ni sorption through laboratory experiments utilizing the birnessite produced by the model bacterium, Pseudomonas putida. Surface excess measurements at pH 6?8 showed that birnessite significantly enhanced Ni sorption at high loadings (up to nearly 4-fold) relative to biomass alone. This apparent large difference in affinity for Ni between the organic and mineral components was confirmed by extended X-ray absorption fine structure spectroscopy, which revealed preferential Ni binding to birnessite cation vacancy sites. At pH >= 7, Ni sorption involved both adsorption and precipitation reactions. Our results thus support the view that the biofilm does not block reactive mineral surface sites; instead, the organic material contributes to metal sorption once high-affinity sites on the mineral are saturated.

Calcineurin A of Candida albicans: involvement in antifungal tolerance, cell morphogenesis and virulence.

The azole antifungal fluconazole possesses only fungistatic activity in Candida albicans and, therefore, this human pathogen is tolerant to this agent. However, tolerance to fluconazole can be inhibited when C. albicans is exposed to fluconazole combined with the immunosuppressive drug cyclosporin A, which is known to inhibit calcineurin activity in yeast. A mutant lacking both alleles of a gene encoding the calcineurin A subunit (CNA) lost viability in the presence of fluconazole, thus making calcineurin essential for fluconazole tolerance. Consistent with this observation, tolerance to fluconazole was modulated by calcium ions or by the expression of a calcineurin A derivative autoactivated by the removal of its C-terminal inhibitory domain. Interestingly, CNA was also essential for tolerance to other antifungal agents (voriconazole, itraconazole, terbinafine, amorolfine) and to several other metabolic inhibitors (caffeine, brefeldin A, mycophenolic acid, fluphenazine) or cell wall-perturbing agents (SDS, calcofluor white, Congo red), thus indicating that the calcineurin pathway plays an important role in the survival of C. albicans in the presence of external growth inhibitors. Several genes, including PMC1, a vacuolar calcium P-type ATPase, were regulated in a calcineurin- and fluconazole-dependent manner. However, PMC1 did not play a direct role in the survival of C. albicans when exposed to fluconazole. In addition to these different properties, calcineurin was found to affect colony morphology in several media known to modulate the C. albicans dimorphic switch. In particular, calcineurin was found to be essential for C. albicans viability in serum-containing media. Finally, calcineurin was found to be necessary for the virulence of C. albicans in a mice model of infection, thus making calcineurin an important element for adequate adaptation to the conditions of the host environment.