Thiamine-auxotrophic mutants of Pseudomonas fluorescens CHA0 are defective in cell-cell signaling and biocontrol factor expression.

In the biocontrol strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to N-acyl-homoserine lactones. Transposon Tn5 was used to isolate P. fluorescens CHA0 insertion mutants that expressed an rsmZ-gfp fusion at reduced levels. Five of these mutants were gacS negative, and in them the gacS mutation could be complemented for exoproduct and signal synthesis by the gacS wild-type allele. Furthermore, two thiamine-auxotrophic (thiC) mutants that exhibited decreased signal synthesis in the presence of 5 x 10(-8) M thiamine were found. Under these conditions, a thiC mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a thiC mutant was impaired for biological control of Pythium ultimum on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by P. fluorescens.

Toll-Interacting Protein Modulates Gut Flora Composition, Epithelial Barrier Integrity and Susceptibility to Colitis

Toll-like receptor ( TLR) s ignals are key to maintaining hostmicrobial i nteractions. T he T oll-interacting-protein (Tollip) is a ubiquitously-expressed inhibitor of inflammasome a nd TLR signaling. W e hypothesized that T ollip might control g ut homeostasis. G enetic ablation of T ollip d id not lead to spontaneous colitis b ut h ad d ramatic c onsequences on t he intestinal expression of the α-defensin cryptidin 4 and the C-type lectin R EGIIIβ. These c hanges were associated with intestinal dysbiosis a nd e nhanced colonization b y segmented filamentous bacteria - a k ey p ro-inflammatory component of the microbiota. Tollip deficiency increased susceptibility to dextran sulfate sodium (DSS) colitis and aggravated chronic Th17-driven colitis in IL-10-/- mice. Flora d epletion w ith a ntibiotics in T ollip-/- mice w as not sufficient to restore DSS colitis susceptibility and deletion of Tollip in n on-hematopoietic c ells using bone-marrow chimeras w as sufficient to increase s usceptibility t o DSS colitis. After D SS administration, we o bserved several e pithelial defects i n Tollip-/- mice including early tight junctions disruption, increased epithelial apoptosis, and increased intestinal permeability. Overall, our data show that T ollip significantly impacts intestinal h omeostasis by controlling b acterial ecology and intestinal r esponse to chemical and immunological stresses.

Pyrroloquinoline quinone biosynthesis gene pqqC, a novel molecular marker for studying the phylogeny and diversity of phosphate-solubilizing pseudomonads.

Many root-colonizing pseudomonads are able to promote plant growth by increasing phosphate availability in soil through solubilization of poorly soluble rock phosphates. The major mechanism of phosphate solubilization by pseudomonads is the secretion of gluconic acid, which requires the enzyme glucose dehydrogenase and its cofactor pyrroloquinoline quinone (PQQ). The main aim of this study was to evaluate whether a PQQ biosynthetic gene is suitable to study the phylogeny of phosphate-solubilizing pseudomonads. To this end, two new primers, which specifically amplify the pqqC gene of the Pseudomonas genus, were designed. pqqC fragments were amplified and sequenced from a Pseudomonas strain collection and from a natural wheat rhizosphere population using cultivation-dependent and cultivation-independent approaches. Phylogenetic trees based on pqqC sequences were compared to trees obtained with the two concatenated housekeeping genes rpoD and gyrB. For both pqqC and rpoD-gyrB, similar main phylogenetic clusters were found. However, in the pqqC but not in the rpoD-gyrB tree, the group of fluorescent pseudomonads producing the antifungal compounds 2,4-diacetylphloroglucinol and pyoluteorin was located outside the Pseudomonas fluorescens group. pqqC sequences from isolated pseudomonads were differently distributed among the identified phylogenetic groups than pqqC sequences derived from the cultivation-independent approach. Comparing pqqC phylogeny and phosphate solubilization activity, we identified one phylogenetic group with high solubilization activity. In summary, we demonstrate that the gene pqqC is a novel molecular marker that can be used complementary to housekeeping genes for studying the diversity and evolution of plant-beneficial pseudomonads.

Phenotypic switching in Pseudomonas brassicacearum involves GacS- and GacA-dependent Rsm small RNAs.

The plant-beneficial bacterium Pseudomonas brassicacearum forms phenotypic variants in vitro as well as in planta during root colonization under natural conditions. Transcriptome analysis of typical phenotypic variants using microarrays containing coding as well as noncoding DNA fragments showed differential expression of several genes relevant to secondary metabolism and of the small RNA (sRNA) genes rsmX, rsmY, and rsmZ. Naturally occurring mutations in the gacS-gacA system accounted for phenotypic switching, which was characterized by downregulation of antifungal secondary metabolites (2,4-diacetylphloroglucinol and cyanide), indoleacetate, exoenzymes (lipase and protease), and three different N-acyl-homoserine lactone molecules. Moreover, in addition to abrogating these biocontrol traits, gacS and gacA mutations resulted in reduced expression of the type VI secretion machinery, alginate biosynthesis, and biofilm formation. In a gacA mutant, the expression of rsmX was completely abolished, unlike that of rsmY and rsmZ. Overexpression of any of the three sRNAs in the gacA mutant overruled the pleiotropic changes and restored the wild-type phenotypes, suggesting functional redundancy of these sRNAs. In conclusion, our data show that phenotypic switching in P. brassicacearum results from mutations in the gacS-gacA system.

Posttranscriptional repression of GacS/GacA-controlled genes by the RNA-binding protein RsmE acting together with RsmA in the biocontrol strain Pseudomonas fluorescens CHA0.

In the plant-beneficial soil bacterium Pseudomonas fluorescens CHA0, the production of biocontrol factors (antifungal secondary metabolites and exoenzymes) is controlled at a posttranscriptional level by the GacS/GacA signal transduction pathway involving RNA-binding protein RsmA as a key regulatory element. This protein is assumed to bind to the ribosome-binding site of target mRNAs and to block their translation. RsmA-mediated repression is relieved at the end of exponential growth by two GacS/GacA-controlled regulatory RNAs RsmY and RsmZ, which bind and sequester the RsmA protein. A gene (rsmE) encoding a 64-amino-acid RsmA homolog was identified and characterized in strain CHA0. Overexpression of rsmE strongly reduced the expression of target genes (hcnA, for a hydrogen cyanide synthase subunit; aprA, for the main exoprotease; and phlA, for a component of 2,4-diacetylphloroglucinol biosynthesis). Single null mutations in either rsmA or rsmE resulted in a slight increase in the expression of hcnA, aprA, and phlA. By contrast, an rsmA rsmE double mutation led to strongly increased and advanced expression of these target genes and completely suppressed a gacS mutation. Both the RsmE and RsmA levels increased with increasing cell population densities in strain CHA0; however, the amount of RsmA showed less variability during growth. Expression of rsmE was controlled positively by GacA and negatively by RsmA and RsmE. Mobility shift assays demonstrated specific binding of RsmE to RsmY and RsmZ RNAs. The transcription and stability of both regulatory RNAs were strongly reduced in the rsmA rsmE double mutant. In conclusion, RsmA and RsmE together account for maximal repression in the GacS/GacA cascade of strain CHA0.

Regulatory RNA as mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens CHA0, an antagonist of root-pathogenic fungi, the GacS/GacA two-component system tightly controls the expression of antifungal secondary metabolites and exoenzymes at a posttranscriptional level, involving the RNA-binding protein and global regulator of secondary metabolism RsmA. This protein was purified from P. fluorescens, and RNA bound to it was converted to cDNA, which served as a probe to isolate the corresponding chromosomal locus, rsmZ. This gene encoded a regulatory RNA of 127 nucleotides and a truncated form lacking 35 nucleotides at the 3' end. Expression of rsmZ depended on GacA, increased with increasing population density, and was stimulated by the addition of a solvent-extractable extracellular signal produced by strain CHA0 at the end of exponential growth. This signal appeared to be unrelated to N-acyl-homoserine lactones. A conserved upstream element in the rsmZ promoter, but not the stress sigma factor RpoS, was involved in rsmZ expression. Overexpression of rsmZ effectively suppressed the negative effect of gacS and gacA mutations on target genes, i.e., hcnA (for hydrogen cyanide synthase) and aprA (for the major exoprotease). Mutational inactivation of rsmZ resulted in reduced expression of these target genes in the presence of added signal. Overexpression of rsmA had a similar, albeit stronger negative effect. These results support a model in which GacA upregulates the expression of regulatory RNAs, such as RsmZ of strain CHA0, in response to a bacterial signal. By a titration effect, RsmZ may then alleviate the repressing activity of RsmA on the expression of target mRNAs.

Invasive mould infections: a multi-disciplinary update.

Systemic fungal infections remain a significant cause of mortality in neutropenic and immunocompromised patients, despite advances in their diagnosis and treatment. The incidence of such infections is rising due to the use of intensive chemotherapy regimens in patients with solid tumours or haematological cancers, the increasing numbers of allogeneic haematopoietic stem cell and solid organ transplants, and the use of potent immunosuppressive therapy in patients with autoimmune disorders. In addition, the epidemiology of systemic fungal infections is changing, with atypical species such as Aspergillus terreus and zygomycetes becoming more common. Treatment has traditionally focused on empirical therapy, but targeted pre-emptive therapy in high-risk patients and prophylactic antifungal treatment are increasingly being adopted. New treatments, including lipid formulations of amphotericin B, second-generation broad-spectrum azoles, and echinocandins, offer effective antifungal activity with improved tolerability compared with older agents; the potential impact of these treatments is reflected in their inclusion in current treatment and prophylaxis guidelines. New treatment strategies, such as aerosolized lipid formulations of amphotericin B, may also reduce the burden of mortality associated with systemic fungal infections. The challenge is to identify ways of coupling potentially effective treatments with early and reliable identification of patients at highest risk of infection.

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.

Biological control of soil-borne pathogens by fluorescent pseudomonads.

Particular bacterial strains in certain natural environments prevent infectious diseases of plant roots. How these bacteria achieve this protection from pathogenic fungi has been analysed in detail in biocontrol strains of fluorescent pseudomonads. During root colonization, these bacteria produce antifungal antibiotics, elicit induced systemic resistance in the host plant or interfere specifically with fungal pathogenicity factors. Before engaging in these activities, biocontrol bacteria go through several regulatory processes at the transcriptional and post-transcriptional levels.

Typing Candida albicans oral isolates from human immunodeficiency virus-infected patients by multilocus enzyme electrophoresis and DNA fingerprinting.

A total of 189 Candida albicans isolates have been typed by multilocus enzyme electrophoresis. The results obtained confirm the clonal mode of reproduction of C. albicans. The C. albicans populations found in the oropharynx of human immunodeficiency virus (HIV)-infected patients, in the oropharynx of healthy carriers, or in association with invasive candidiasis could not be distinguished. No clone or group of clones could be associated with the appearance of clinical disorders or with a reduced in vitro susceptibility to the antifungal agent fluconazole. Multiple and sequential oral isolates from 24 HIV-infected patients were also typed by restriction enzyme analysis with the enzymes EcoRI and HinfI and by use of the Ca3 repetitive probe. The results obtained by the combination of all three typing methods show that all but one patient each carried a unique major C. albicans clone in their oropharynx. The 21 patients with sequential isolates had the same C. albicans clones in their throats during recurrent oropharyngeal candidiasis episodes, independently of clinical status or of changes of in vitro susceptibility to fluconazole. Finally, several isolates of the same C. albicans clone found simultaneously in the oropharynx of a patient may present different levels of susceptibility to fluconazole.

Candida colonization index and subsequent infection in critically ill surgical patients: 20 years later.

INTRODUCTION: For decades, clinicians dealing with immunocompromised and critically ill patients have perceived a link between Candida colonization and subsequent infection. However, the pathophysiological progression from colonization to infection was clearly established only through the formal description of the colonization index (CI) in critically ill patients. Unfortunately, the literature reflects intense confusion about the pathophysiology of invasive candidiasis and specific associated risk factors. METHODS: We review the contribution of the CI in the field of Candida infection and its development in the 20 years following its original description in 1994. The development of the CI enabled an improved understanding of the pathogenesis of invasive candidiasis and the use of targeted empirical antifungal therapy in subgroups of patients at increased risk for infection. RESULTS: The recognition of specific characteristics among underlying conditions, such as neutropenia, solid organ transplantation, and surgical and nonsurgical critical illness, has enabled the description of distinct epidemiological patterns in the development of invasive candidiasis. CONCLUSIONS: Despite its limited bedside practicality and before confirmation of potentially more accurate predictors, such as specific biomarkers, the CI remains an important way to characterize the dynamics of colonization, which increases early in patients who develop invasive candidiasis.

Treatment and timing in invasive mould disease.

Invasive mould disease is a growing threat for immunocompromised patients. The optimum time to use mould-active antifungal agents is much debated. Current approaches to antifungal prophylaxis, early treatment (empirical and pre-emptive therapy) and treatment of documented mould infections in onco-haematology patients are discussed.

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.

Invasive Candida infections in the ICU

Invasive candidiasis, including candidemia and deep-seated Candida infections, is a severe opportunistic infection with an overall mortality in ICU patients comparable to that of severe sepsis/septic shock. With an incidence ranging from 5 to 10 cases per 1000 ICU admissions, invasive candidiasis represents 510% of all ICU-acquired infections. Although a high proportion of critically ill patients is colonised with Candida spp., only 540% develop an invasive infection. The occurrence of this complication is difficult to predict and an early diagnosis remains a major challenge. Indeed, blood cultures are positive in a minority of cases and often late in the course of infection. New non-culture based laboratory techniques may contribute to early diagnosis and management of invasive candidiasis. Recent data suggest that prediction rules based on risk factors, clinical and microbiological parameters or monitoring of Candida colonisation may efficiently identify critically ill patients at high risk of invasive candidiasis who may benefit of preventive or pre-emptive antifungal therapy. In many cancer centres, exposure to azoles antifungals has been associated with an epidemiological shift from Candida albicans to non-albicans Candida species with reduced antifungal susceptibility or intrinsic resistance. This trend has not been observed in recent surveys on candidemia in non-immunocompromised ICU patients. Prophylaxis, pre-emptive or empirical antifungal treatment are possible approaches for prevention or early management of invasive candidiasis. However, the selection of high-risk patients remains critical for an efficient management aimed at reducing the number needed to treat and thus avoiding unnecessary treatments associated with the emergence of resistance, drug toxicity and costs.