Caspofungin at sub-inhibitory concentration promotes the formation of Candida albicans persister cells.

AIMS: Low caspofungin exposure is frequently encountered in patients with invasive candidiasis caused by Candida albicans. This study aimed to investigate the effects of caspofungin on C. albicans at sub-inhibitory concentrations. METHODS AND RESULTS: First, a comparative transcriptomics analysis was performed on C. albicans receiving caspofungin at sub-minimum inhibitory concentrations (sub-MICs). The results showed that caspofungin significantly changed the mRNA expression profile in DAY185, with DE-mRNAs enriched in the functions of cell wall biosynthesis, metabolism, etc. Subsequently, cellular fitness, cell aggregation, energy metabolism activity and the proportion of persister cells of C. albicans were quantitatively and/or qualitatively assessed after sub-MIC caspofungin exposure. No significant changes in cell fitness and aggregation formation were observed during treatment of C. albicans with sub-MIC caspofungin. In C. albicans aggregation treated with sub-MIC caspofungin, we observed a decrease in respiratory metabolism and an increase in persister cells; this effect was more pronounced in als1ΔΔ than in DAY185. CONCLUSIONS: Pre-exposure to sub-MIC caspofungin suppresses C. albicans respiratory metabolism and promotes persister cell development. SIGNIFICANCE AND IMPACT OF THE STUDY: Caspofungin should be used with caution in patients with C. albicans infections, as anti-infection therapy may fail due to persister cells.

Modulation of Cyp450, ALS1 and COX-2 signaling pathways induced by Candida albicans infection via novel antifungal agents.

Although, fluconazole is widely used in clinical treatment as an antifungal drug, it recorded potential problems as resistance and intracellular accumulation. Female albino mice were injected with single ip dose of Candida albicans (1.5 × 106 CFU). Three weeks post treatment with fluconazole and two novel synthesized compounds [(2-(4-(Pyridin-2-yl) aminosulfonylphenylamino)-6-(naphthalen-2-yl)-4-(pyridin-2-yl) pyridine-3carbonitrile) and (2-(4-(Pyrimidin-2-yl) aminosulfonylphenylamino)-6-(naphthalen-2-yl)-4-(pyridine-2-yl)pyridine-3-carbonitrile) (13b & 14b, respectively)] in both low and high doses (50 mg/kg & 200 mg/kg), liver function and vaginal inflammation were assessed. Candida albicans significantly elevated serum alanine aminotransferase (ALT) and butrylcholinesterase (BCHE) as well as hepatic malondialdehyde (MDA). Molecular analysis confirmed a significant up-regulation in mRNA gene expression of Agglutinin-like sequence (ALS1), hepatic cytochrome p450 (Cyp450). Vaginal COX-2 gene expression was also elevated. Nevertheless, a significant down-regulation was apparent in mice treated with the aforementioned compounds. Meanwhile, administration of 14b in a high dose noticeably down-regulated the altered parameters expression showing a significant effect in comparison to animals treated with the variable doses of the tested compounds. Histopathological finding confirmed the obtained results. The current work investigated the efficiency of new synthetic pyrimidine derivatives 14bas anti-microbial agents and recommended to be improved and evaluated as a novel antifungal drug to overcome the emergence of resistance problem.

Disruption of the rice ALS1 localized in chloroplast causes seedling-lethal albino phenotype.

Chloroplasts are the organelles responsible for photosynthesis and regulate normal plant growth. Although translation elongation factors play important roles in chloroplast development, functional studies of chloroplast translation elongation factors in higher plants remain very sparse. Here, we obtained a rice mutant exhibiting seedling-lethal albino phenotype and named it albino and lethal seedling 1 (als1). Consistently, low content of photosynthetic pigments, malformed chloroplasts and defective photosynthesis were observed in als1 mutant leaves. Map-based cloning experiment showed that als1 mutant had a T base insertion in Os02g0595700, causing a frame shift and premature stop codon. ALS1 encoded a GTP-binding protein EF-Tu, which acts as a translation elongation factor in chloroplast protein translation. ALS1 was found to be expressed throughout plant with highest expression level in young leaves. Moreover, ALS1 was located in chloroplast, whereas the truncated als1 could not normally be located in chloroplast. Additionally, the ALS1 mutation significantly influenced the expression of downstream genes, such as genes relevant to chlorophyll biosynthesis, photosynthesis as well as chloroplast development. These results show that ALS1 acts as a key regulator of chloroplast development and plant growth.

BDSF inhibits Candida albicans adherence to urinary catheters.

Cis-2-dodecenoic acid (BDSF) is a quorum-sensing signal molecule produced by the opportunistic pathogen Burkholderia cenocepacia and suppresses germ tube formation of Candida albicans. An in vitro model for biofilm formation evaluated the influence of BDSF on C. albicans. Biofilm morphology was observed using scanning electron microscopy, cell adherence was determined using polystyrene plates and siliconized urinary catheters, and the levels of expression of genes involved in adhesion were determined using Real-time Reverse-Transcriptase Polymerase Chain Reaction. BDSF inhibited initial biofilm formation by a clinical isolate of C. albicans and reduced its capability to adhere to the polystyrene surface. BDSF at concentrations up to 120 µM did not significantly affect the viability of C. albicans. BDSF (90 µM) inhibited cell adherence to plates and catheters by 4- and 25-fold. Compared with untreated yeasts, the level of expression of genes involved in adhesion, ALS1 and EAP1, were reduced by 4- and 0.25-fold, whereas that of YWP1 was increased at a 4-fold higher level. Here we show that BDSF effectively inhibited biofilm development as indicated by its ability to inhibit adherence. Thus, BDSF should be considered as a potential therapeutic agent to prevent disease caused by Candida species.

Candida albicans Adhesins Als1 and Hwp1 Modulate Interactions with Streptococcus mutans.

Candida albicans and Streptococcus mutans are known to synergistically interact with each other in the oral cavity. For example, glucosyltransferase B (GtfB), secreted by S. mutans, can bind to the C. albicans cell surface, promoting dual-species biofilm formation. However, the fungal factors mediating interactions with S. mutans are unknown. The C. albicans adhesins Als1, Als3, and Hwp1 are key players in C. albicans single-species biofilm formation, but their roles, if any, in interacting with S. mutans have not been assessed. Here, we investigated the roles of the C. albicans cell wall adhesins Als1, Als3, and Hwp1 on forming dual-species biofilms with S. mutans. We assessed the abilities of the C. albicans wild-type als1Δ/Δ, als3Δ/Δ, als1Δ/Δ/als3Δ/Δ, and hwp1Δ/Δ strains to form dual-species biofilms with S. mutans by measuring optical density, metabolic activity, cell enumeration, biomass, thickness, and architecture of the biofilms. We observed that the C. albicans wild-type strain formed enhanced dual-species biofilms in the presence of S. mutans in these different biofilm assays, confirming that C. albicans and S. mutans synergistically interact in the context of biofilms. Our results reveal that C. albicans Als1 and Hwp1 are major players in interacting with S. mutans, since dual-species biofilm formation was not enhanced when the als1Δ/Δ or hwp1Δ/Δ strains were cultured with S. mutans in dual-species biofilms. Als3, however, does not seem to play a clear role in interacting with S. mutans in dual-species biofilm formation. Overall, our data suggest that the C. albicans adhesins Als1 and Hwp1 function to modulate interactions with S. mutans and could be potential targets for future therapeutics.

Alteration of Mitochondrial Integrity as Upstream Event in the Pathophysiology of SOD1-ALS.

Little is known about the early pathogenic events by which mutant superoxide dismutase 1 (SOD1) causes amyotrophic lateral sclerosis (ALS). This lack of mechanistic understanding is a major barrier to the development and evaluation of efficient therapies. Although protein aggregation is known to be involved, it is not understood how mutant SOD1 causes degeneration of motoneurons (MNs). Previous research has relied heavily on the overexpression of mutant SOD1, but the clinical relevance of SOD1 overexpression models remains questionable. We used a human induced pluripotent stem cell (iPSC) model of spinal MNs and three different endogenous ALS-associated SOD1 mutations (D90Ahom, R115Ghet or A4Vhet) to investigate early cellular disturbances in MNs. Although enhanced misfolding and aggregation of SOD1 was induced by proteasome inhibition, it was not affected by activation of the stress granule pathway. Interestingly, we identified loss of mitochondrial, but not lysosomal, integrity as the earliest common pathological phenotype, which preceded elevated levels of insoluble, aggregated SOD1. A super-elongated mitochondrial morphology with impaired inner mitochondrial membrane potential was a unifying feature in mutant SOD1 iPSC-derived MNs. Impaired mitochondrial integrity was most prominent in mutant D90Ahom MNs, whereas both soluble disordered and detergent-resistant misfolded SOD1 was more prominent in R115Ghet and A4Vhet mutant lines. Taking advantage of patient-specific models of SOD1-ALS in vitro, our data suggest that mitochondrial dysfunction is one of the first crucial steps in the pathogenic cascade that leads to SOD1-ALS and also highlights the need for individualized medical approaches for SOD1-ALS.

Semi-dominant mutation in the cysteine-rich receptor-like kinase gene, ALS1, conducts constitutive defence response in rice.

Plants have evolved a sophisticated two-branch defence system to prevent the growth and spread of pathogen infection. The novel Cys-rich repeat (CRR) containing receptor-like kinases, known as CRKs, were reported to mediate defence resistance in plants. For rice, there are only two reports of CRKs. A semi-dominant lesion mimic mutant als1 (apoptosis leaf and sheath 1) in rice was identified to demonstrate spontaneous lesions on the leaf blade and sheath. A map-based cloning strategy was used for fine mapping and cloning of ALS1, which was confirmed to be a typical CRK in rice. Functional studies of ALS1 were conducted, including phylogenetic analysis, expression analysis, subcellular location and blast resistance identification. Most pathogenesis-related (PR) genes and other defence-related genes were activated and up-regulated to a high degree. ALS1 was expressed mainly in the leaf blade and sheath, in which further study revealed that ALS1 was present in the vascular bundles. ALS1 was located in the cell membrane of rice protoplasts, and its mutation did not change its subcellular location. Jasmonic acid (JA) and salicylic acid (SA) accumulation were observed in als1, and enhanced blast resistance was also observed. The mutation of ALS1 caused a constitutively activated defence response in als1. The results of our study imply that ALS1 participates in a defence response resembling the common SA-, JA- and NH1-mediated defence responses in rice.

Haplotype Analysis of the First A4V-SOD1 Spanish Family: Two Separate Founders or a Single Common Founder?

Despite the genetic heterogeneity reported in familial amyotrophic lateral sclerosis (ALS) (fALS), Cu/Zn superoxide-dismutase (SOD1) gene mutations are the second most common cause of the disease, accounting for around 20% of all families (ALS1) and isolated sporadic cases (sALS). At least 186 different mutations in the SOD1 gene have been reported to date. The possibility of a single founder and separate founders have been investigated for D90A (p.D91A) and A4V (p.A5V), the most common mutations worldwide. High-throughput single nucleotide polymorphism genotyping studies have suggested two founders for A4V (one for the Amerindian population and another for the European population) although the possibility that the two populations are descended from a single ancient founder cannot be ruled out. We used 15 genetic variants spanning the human chromosome 21 from the SOD1 gene to the SCAF4 gene, comparing them with the population reference panels, to demonstrate that the first A4V Spanish pedigree shared the genetic background reported in the European population.

S. oralis activates the Efg1 filamentation pathway in C. albicans to promote cross-kingdom interactions and mucosal biofilms.

Candida albicans and Streptococcus oralis are ubiquitous oral commensal organisms. Under host-permissive conditions these organisms can form hypervirulent mucosal biofilms. C. albicans biofilm formation is controlled by 6 master transcriptional regulators: Bcr1, Brg1, Efg1, Tec1, Ndt80, and Rob1. The objective of this work was to test whether any of these regulators play a role in cross-kingdom interactions between C. albicans and S. oralis in oral mucosal biofilms, and identify downstream target gene(s) that promote these interactions. Organotypic mucosal constructs and a mouse model of oropharyngeal infection were used to analyze mucosal biofilm growth and fungal gene expression. By screening 6 C. albicans transcription regulator reporter strains we discovered that EFG1 was strongly activated by interaction with S. oralis in late biofilm growth stages. EFG1 gene expression was increased in polymicrobial biofilms on abiotic surfaces, mucosal constructs and tongue tissues of mice infected with both organisms. EFG1 was required for robust Candida-streptococcal biofilm growth in organotypic constructs and mouse oral tissues. S. oralis stimulated C. albicans ALS1 gene expression in an EFG1-dependent manner, and Als1 was identified as a downstream effector of the Efg1 pathway which promoted C. albicans-S. oralis coaggregation interactions in mixed biofilms. We conclude that S. oralis induces an increase in EFG1 expression in C. albicans in late biofilm stages. This in turn increases expression of ALS1, which promotes coaggregation interactions and mucosal biofilm growth. Our work provides novel insights on C. albicans genes which play a role in cross-kingdom interactions with S. oralis in mucosal biofilms.

Inhibition of Candida albicans biofilm development by unencapsulated Enterococcus faecalis cps2.

BACKGROUND/PURPOSE: In the oral environment, Candida albicans interacts with many bacteria, including Enterococcus faecalis. We investigated the susceptibility of C. albicans biofilm development to the presence of unencapsulated E. faecalis cps2 in comparison with reference strains (E. faecalis ATCC 29212) or their respective spent medium (collected at 6 hours). MATERIAL AND METHODS: Crystal violet stain was used to measure the total biofilm mass, whereas quantitative real-time polymerase chain reaction was used to analyze the change in expression of the mRNA of hypha morphology (ALS1 and ALS3) and biofilm maturation (EFB1). RESULTS: At the intermediate stage, C. albicans resisted the presence of each E. faecalis strain tested and their spent medium. However, at the maturation stage, the unencapsulated strain was stronger in reducing C. albicans biofilms than the reference strain (P < 0.05). At this maturation stage, the transcription levels of each gene tested decreased in the presence of either E. faecalis strains or their respective spent medium. The unencapsulated strain was more pronounced in reducing ALS1/ALS3 expression, whereas the respective spent medium had a similar capability to restrict the expression of EFB1. CONCLUSION: This study showed, the unencapsulated strain is more effective in inhibiting C. albicans biofilm development compared with the reference strains. In contrast, the secreted molecules produced by each strain tested are necessary in controlling the growths of C. albicans biofilm.

Differential activation of genes related to aluminium tolerance in two contrasting rice cultivars.

Rice (Oryza sativa) is a highly Al-tolerant crop. Among other mechanisms, a higher expression of STAR1/STAR2 (sensitive to Al rhizotoxicity1/2) genes and of Nrat1 (NRAMP Aluminium Transporter 1), and ALS1 (Aluminium sensitive 1) can at least in part be responsible for the inducible Al tolerance in this species. Here we analysed the responses to Al in two contrasting rice varieties. All analysed toxicity/tolerance markers (root elongation, Evans blue, morin and haematoxylin staining) indicated higher Al-tolerance in variety Nipponbare, than in variety Modan. Nipponbare accumulated much less Al in the roots than Modan. Aluminium supply caused stronger expression of STAR1 in Nipponbare than in Modan. A distinctively higher increase of Al-induced abscisic acid (ABA) accumulation was found in the roots of Nipponbare than in Modan. Highest ABA levels were observed in Nipponbare after 48 h exposure to Al. This ABA peak was coincident in time with the highest expression level of STAR1. It is proposed that ABA may be required for cell wall remodulation facilitated by the enhanced UDP-glucose transport to the walls through STAR1/STAR2. Contrastingly, in the roots of Modan the expression of both Nrat1 coding for a plasma membrane Al-transporter and of ALS1 coding for a tonoplast-localized Al transporter was considerably enhanced. Moreover, Modan had a higher Al-induced expression of ASR1 a gene that has been proposed to code for a reactive oxygen scavenging protein. In conclusion, the Al-exclusion strategy of Nipponbare, at least in part mediated by STAR1 and probably regulated by ABA, provided better protection against Al toxicity than the accumulation and internal detoxification strategy of Modan mediated by Nrat1, ALS1 and ARS1.