dThe Candida haemulonii species complex is currently known as C. haemulonii groups I and II. Here we describe C. haemulonii group II as a new species, Candida duobushaemulonii sp. nov., and C. haemulonii var. vulnera as new a variety of C. haemulonii group I using phenotypic and molecular methods. These taxa and other relatives of C. haemulonii (i.e., Candida auris and Candida pseudohaemulonii) cannot be differentiated by the commercial methods now used for yeast identification. Four isolates (C. haemulonii var. vulnera) differed from the other isolates of C. haemulonii in the sequence of the internal transcribed spacer (ITS) regions of the nuclear rRNA gene operon. The new species and the new variety have a multiresistant antifungal profile, which includes high MICs of amphotericin B (geometric mean MIC, 1.18 mg/liter for C. haemulonii var. vulnera and 2 mg/liter for C. duobushaemulonii sp. nov) and cross-resistance to azole compounds. Identification of these species should be based on molecular methods, such as sequence analysis of ITS regions and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Candida and Aspergillus species are the most common causes of invasive fungal infections in immunocompromised individuals, but besides these fungi, many other yeast species and filamentous fungi can be pathogenic in such individuals (7). The list of reported species that cause human infection is constantly growing, partly because of recent advances in molecular tools and diagnostics. Thus, new clinically relevant species such as Candida metapsilosis, Candida orthopsilosis, Candida bracariensis, and Candida nivariensis, have been described recently (1,5,39).Candida haemulonii (van Uden and Kolipinsky) S. A. Meyer and D. Yarrow (41) (syn. Torulopsis haemulonii) is one of the rare yeast species that can be isolated from human clinical sources. The species originally described was from the gut of a blue-striped grunt fish (Haemulon scirus) in 1962 (40). The first isolation of this yeast from a human, i.e., from the blood of a patient with renal failure, was reported by Lavarde et al. (22). Since then, several cases of infections due to this yeast have been described in the literature, varying from superficial to deep infections. Cases of catheter-related fungemia (18), bloodstream infections (30,34), and osteitis (6) and outbreaks in intensive care units (16) have been reported recently. The species has also been isolated from toenails of diabetic patients (13). Noteworthy is the susceptibility profile of this yeast, which shows high MICs of amphotericin B (AMB) and fluconazole (FLC) (ranges, 0.5 to 32 and 4 to Ͼ64 mg/liter, respectively), which can hinder the management of patients with deep infections caused by this yeast. This antifungal profile has often been associated with clinical failure (6,16,17,30,34).The C. haemulonii species complex was further studied by Lehman et al. in 1993 (24). They studied 25 strains from different geographic origins and clinical sources and described two gene...

Under a shortage of oxygen, bacterial growth can be faced mainly by two ATP-generating mechanisms: (i) by synthesis of specific high-affinity terminal oxidases that allow bacteria to use traces of oxygen or (ii) by utilizing other substrates as final electron acceptors such as nitrate, which can be reduced to dinitrogen gas through denitrification or to ammonium. This bacterial respiratory shift from oxic to microoxic and anoxic conditions requires a regulatory strategy which ensures that cells can sense and respond to changes in oxygen tension and to the availability of other electron acceptors. Bacteria can sense oxygen by direct interaction of this molecule with a membrane protein receptor (e.g., FixL) or by interaction with a cytoplasmic transcriptional factor (e.g., Fnr). A third type of oxygen perception is based on sensing changes in redox state of molecules within the cell. Redox-responsive regulatory systems (e.g., ArcBA, RegBA/PrrBA, RoxSR, RegSR, ActSR, ResDE, and Rex) integrate the response to multiple signals (e.g., ubiquinone, menaquinone, redox active cysteine, electron transport to terminal oxidases, and NAD/NADH) and activate or repress target genes to coordinate the adaptation of bacterial respiration from oxic to anoxic conditions. Here, we provide a compilation of the current knowledge about proteins and regulatory networks involved in the redox control of the respiratory adaptation of different bacterial species to microxic and anoxic environments.

Summary A role for specific natural products in directly mediating antagonistic plant–plant interactions –that is, allelopathy –has been controversial. If proven, such phenomena would hold considerable promise for agronomic improvement of staple food crops such as rice (Oryza sativa).However, while substantiated by the presence of phytotoxic compounds at potentially relevant levels, demonstrating a direct role for specific natural products in allelopathy has been difficult due to the chemical complexity of root and plant litter exudates. This complexity can be bypassed via selective genetic manipulation to ablate production of putative allelopathic compounds, but such an approach previously has not been applied.The rice diterpenoid momilactones provide an example of natural products for which correlative biochemical evidence has been obtained for a role in allelopathy. Here, we apply reverse genetics, using knock-outs of the relevant diterpene synthases (OsCPS4 and OsKSL4), to demonstrate that rice momilactones are involved in allelopathy, including suppressing growth of the widespread rice paddy weed, barnyard grass (Echinochloa crus-galli).Thus, our results not only provide novel genetic evidence for natural product mediated allelopathy, but also furnish a molecular target for breeding and metabolic engineering of this important crop plant.

RNA-binding proteins play a central role in post-transcriptional mechanisms that control gene expression. Identification of novel RNA-binding proteins in fungi is essential to unravel post-transcriptional networks and cellular processes that confer identity to the fungal kingdom. Here, we carried out the functional characterisation of the filamentous fungus-specific RNA-binding protein RBP35 required for full virulence and development in the rice blast fungus. RBP35 contains an N-terminal RNA recognition motif (RRM) and six Arg-Gly-Gly tripeptide repeats. Immunoblots identified two RBP35 protein isoforms that show a steady-state nuclear localisation and bind RNA in vitro. RBP35 coimmunoprecipitates in vivo with Cleavage Factor I (CFI) 25 kDa, a highly conserved protein involved in polyA site recognition and cleavage of pre-mRNAs. Several targets of RBP35 have been identified using transcriptomics including 14-3-3 pre-mRNA, an important integrator of environmental signals. In Magnaporthe oryzae, RBP35 is not essential for viability but regulates the length of 3′UTRs of transcripts with developmental and virulence-associated functions. The Δrbp35 mutant is affected in the TOR (target of rapamycin) signaling pathway showing significant changes in nitrogen metabolism and protein secretion. The lack of clear RBP35 orthologues in yeast, plants and animals indicates that RBP35 is a novel auxiliary protein of the polyadenylation machinery of filamentous fungi. Our data demonstrate that RBP35 is the fungal equivalent of metazoan CFI 68 kDa and suggest the existence of 3′end processing mechanisms exclusive to the fungal kingdom.

Background The clinical presentation of COVID-19 in patients admitted to hospital is heterogeneous. We aimed to determine whether clinical phenotypes of patients with COVID-19 can be derived from clinical data, to assess the reproducibility of these phenotypes and correlation with prognosis, and to derive and validate a simplified probabilistic model for phenotype assignment. Phenotype identification was not primarily intended as a predictive tool for mortality. MethodsIn this study, we used data from two cohorts: the COVID-19@Spain cohort, a retrospective cohort including 4035 consecutive adult patients admitted to 127 hospitals in Spain with COVID-19 between Feb 2 and March 17, 2020, and the COVID-19@HULP cohort, including 2226 consecutive adult patients admitted to a teaching hospital in Madrid between Feb 25 and April 19, 2020. The COVID-19@Spain cohort was divided into a derivation cohort, comprising 2667 randomly selected patients, and an internal validation cohort, comprising the remaining 1368 patients. The COVID-19@HULP cohort was used as an external validation cohort. A probabilistic model for phenotype assignment was derived in the derivation cohort using multinomial logistic regression and validated in the internal validation cohort. The model was also applied to the external validation cohort. 30-day mortality and other prognostic variables were assessed in the derived phenotypes and in the phenotypes assigned by the probabilistic model. Findings Three distinct phenotypes were derived in the derivation cohort (n=2667)-phenotype A (516 [19%] patients), phenotype B (1955 [73%]) and phenotype C (196 [7%])-and reproduced in the internal validation cohort (n=1368)phenotype A (233 [17%] patients), phenotype B (1019 [74%]), and phenotype C (116 [8%]). Patients with phenotype A were younger, were less frequently male, had mild viral symptoms, and had normal inflammatory parameters. Patients with phenotype B included more patients with obesity, lymphocytopenia, and moderately elevated inflammatory parameters. Patients with phenotype C included older patients with more comorbidities and even higher inflammatory parameters than phenotype B. We developed a simplified probabilistic model (validated in the internal validation cohort) for phenotype assignment, including 16 variables. In the derivation cohort, 30-day mortality rates were 2•5% (95% CI 1•4-4•3) for patients with phenotype A, 30•5% (28•5-32•6) for patients with phenotype B, and 60•7% (53•7-67•2) for patients with phenotype C (log-rank test p<0•0001). The predicted phenotypes in the internal validation cohort and external validation cohort showed similar mortality rates to the assigned phenotypes (internal validation cohort: 5•3% [95% CI 3•4-8•1] for phenotype A, 31•3% [28•5-34•2] for phenotype B, and 59•5% [48•8-69•3] for phenotype C; external validation cohort: 3•7% [2•0-6•4] for phenotype A, 23•7% [21•8-25•7] for phenotype B, and 51•4% [41•9-60•7] for phenotype C).Interpretation Patients admitted to hospital with COVID-19 can be classified into three...

It is becoming recognized that leghaemoglobin constitutes an important buffer for the cytotoxic nitric oxide radical (NO N ) in root nodules, although the sources of this NO N within nodules are unclear. In Bradyrhizobium japonicum bacteroids, NO N can be produced through the denitrification process, during which nitrate is reduced to nitrite by the periplasmic nitrate reductase Nap, and nitrite is reduced to NO N by the respiratory nitrite reductase NirK. To assess the contribution of bacteroidal denitrification to the NO N within nitrate-treated soybean nodules, electron paramagnetic resonance and UV-visible spectroscopy were employed to study the presence of nitrosylleghaemoglobin (LbNO) within nodules from plants inoculated with wild-type, napA or nirK B. japonicum strains. Since it has been found that hypoxia induces NO N production in plant root tissue, and that plant roots can be subjected to hypoxic stress during drought and flooding, the effect of hypoxic stress on the formation of LbNO complexes within nodules was also investigated. Maximal levels of LbNO were observed in nodules from plants treated with nitrate and subjected to hypoxic conditions. It is shown that, in the presence of nitrate, all of the LbNO within normoxic nodules arises from nitrate reduction by the bacteroidal periplasmic nitrate reductase, whereas Nap activity is only responsible for half of the LbNO within hypoxic nodules. In contrast to Nap, NirK is not essential for LbNO formation under any condition tested.

To survive and proliferate in the absence of oxygen, many enteric pathogens can undergo anaerobic respiration within the host by using nitrate (NO 3 - ) as electron acceptor 1 , 2 . In these bacteria, NO 3 - is typically reduced by a nitrate reductase to nitrite (NO 2 - ), a toxic intermediate that is further reduced by a nitrite reductase 3 . However, Vibrio cholerae, the intestinal pathogen that causes cholera, lacks a nitrite reductase, leading to NO 2 - accumulation during nitrate reduction 4 . Thus, V. cholerae is thought to be unable to undergo NO 3 - -dependent anaerobic respiration 4 . Here, we show that during hypoxic growth, NO 3 - reduction in V. cholerae divergently impacts bacterial fitness in a manner dependent on environmental pH. Remarkably, in alkaline conditions, V. cholerae can reduce NO 3 - to support population growth. Conversely, in acidic conditions, accumulation of NO 2 - from NO 3 - reduction simultaneously limits population expansion and preserves cell viability by lowering fermentative acid production. Interestingly, other bacterial species such as Salmonella typhimurium, enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium also reproduced this pH-dependent response suggesting that this mechanism might be conserved within enteric pathogens. Our findings explain how a bacterial pathogen can use a single redox reaction to divergently regulate population expansion depending on fluctuating environmental pH.

Recombinant attenuated Salmonella enterica serovar Typhimurium strains are believed to act as powerful live vaccine carriers that are able to elicit protection against various pathogens. Auxotrophic mutations, such as a deletion of aroA, are commonly introduced into such bacteria for attenuation without incapacitating immunostimulation. In this study, we describe the surprising finding that deletion of aroA dramatically increased the virulence of attenuated Salmonella in mouse models. Mutant bacteria lacking aroA elicited increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) after systemic application. A detailed genetic and phenotypic characterization in combination with transcriptomic and metabolic profiling demonstrated that ΔaroA mutants display pleiotropic alterations in cellular physiology and lipid and amino acid metabolism, as well as increased sensitivity to penicillin, complement, and phagocytic uptake. In concert with other immunomodulating mutations, deletion of aroA affected flagellin phase variation and gene expression of the virulence-associated genes arnT and ansB. Finally, ΔaroA strains displayed significantly improved tumor therapeutic activity. These results highlight the importance of a functional shikimate pathway to control homeostatic bacterial physiology. They further highlight the great potential of ΔaroA-attenuated Salmonella for the development of vaccines and cancer therapies with important implications for host-pathogen interactions and translational medicine.