- Volume 3, Issue 12, 2021

Objectives. Challenges remain and there are still a sufficient number of cases with epidemiological, clinical features and radiological data suggestive of COVID-19 pneumonia that persist negative in their RT-PCR results. The aim of the study was to define the distinguishing characteristics between patients developing a serological response to SARS-CoV-2 and those who did not.

Methods. RT-PCR tests used were TaqPath 2019-nCoV Assay Kit v1 (ORF-1ab, N and S genes) from Thermo Fisher Diagnostics and SARS-COV-2 Kit (N and E genes) from Vircell. Serological response was tested using the rapid SARS-CoV2 IgG/IgM Test Cassette from T and D Diagnostics Canada and CMC Medical Devices and Drugs, S.L, CE.

Results. In this cross-sectional study, we included a cohort of 52 patients recruited from 31 March 2020 to 23 April 2020. Patients with positive serology had an older average age (73.29) compared to those who were negative (54.82) (P<0.05). Sat02 in 27 of 34 patients with positive serology were below 94% (P<0.05). There was a frequency of 1.5% negative SARS-CoV-2 RT-PCRs during the study period concurring with 36.7% of positivity.

Conclusions. Clinical features and other biomarkers in a context of a positive serology can be considered crucial for diagnosis.

Five hundred and thirty-four unrelated Pseudomonas aeruginosa isolates from inanimate habitats, patients with cystic fibrosis (CF) and other human infections were sequenced in 19 genes that had been identified previously as the hot spots of genomic within-host evolution in serial isolates from 12 CF lungs. Amplicon sequencing confirmed a significantly higher sequence diversity of the 19 loci in P. aeruginosa isolates from CF patients compared to those from other habitats, but this overrepresentation was mainly due to the larger share of synonymous substitutions. Correspondingly, non-synonymous substitutions were either rare (gltT, lepA, ptsP) or benign (nuoL, fleR, pelF) in some loci. Other loci, however, showed an accumulation of non-neutral coding variants. Strains from the CF habitat were often mutated at evolutionarily conserved positions in the elements of stringent response (RelA, SpoT), LPS (PagL), polyamine transport (SpuE, SpuF) and alginate biosynthesis (AlgG, AlgU). The strongest skew towards the CF lung habitat was seen for amino acid sequence variants in AlgG that clustered in the carbohydrate-binding/sugar hydrolysis domain. The master regulators of quorum sensing lasR and rhlR were frequent targets for coding variants in isolates from chronic and acute human infections. Unique variants in lasR showed strong evidence of positive selection indicated by d N/d S values of ~4. The pelA gene that encodes a multidomain enzyme involved in both the formation and dispersion of Pel biofilms carried the highest number of single-nucleotide variants among the 19 genes and was the only gene with a higher frequency of missense mutations in P. aeruginosa strains from non-CF habitats than in isolates from CF airways. PelA protein variants are widely distributed in the P. aeruginosa population. In conclusion, coding variants in a subset of the examined loci are indeed characteristic for the adaptation of P. aeruginosa to the CF airways, but for other loci the elevated mutation rate is more indicative of infections in human habitats (lasR, rhlR) or global diversifying selection (pelA).

Some major producers of useful bioactive natural products belong to the genus Streptomyces or related actinobacteria. Genetic engineering of these bacteria and the pathways that synthesize their valuable products often relies on serine integrases. To further improve the flexibility and efficiency of genome engineering via serine integrases, we explored whether multiple integrating vectors encoding orthogonally active serine integrases can be introduced simultaneously into Streptomyces recipients via conjugal transfer and integration. Pairwise combinations of Escherichia coli donors containing vectors encoding orthogonal serine integrases were used in each conjugation. Using donors containing plasmids (of various sizes) encoding either the φBT1 or the φC31 integration systems, we observed reproducible simultaneous plasmid integration into Streptomyces coelicolor and Streptomyces lividans at moderate frequencies after conjugation. This work demonstrated how site-specific recombination based on orthogonal serine integrases can save researchers time in genome engineering experiments in Streptomyces .

The discovery and adaptation of CRISPR/Cas systems within molecular biology has provided advances across biological research, agriculture and human health. Genomic manipulation through use of a CRISPR nuclease and programmed guide RNAs has become a common and widely accessible practice. The identification and introduction of new engineered variants and orthologues of Cas9 as well as alternative CRISPR systems such as the type V group have provided additional molecular options for editing. These include distinct PAM requirements, staggered DNA double-strand break formation, and the ability to multiplex guide RNAs from a single expression construct. Use of CRISPR/Cas has allowed for the construction and testing of a powerful genetic architecture known as a gene drive within eukaryotic model systems. Our previous work developed a drive within budding yeast using Streptococcus pyogenes Cas9. Here, we installed the type V Francisella novicida Cas12a (Cpf1) nuclease gene and its corresponding guide RNA to power a highly efficient artificial gene drive in diploid yeast. We examined the consequence of altering guide length or introduction of individual mutational substitutions to the crRNA sequence. Cas12a-dependent gene-drive function required a guide RNA of at least 18 bp and could not tolerate most changes within the 5′ end of the crRNA.

The global use of organophosphate insecticides (OPPs) and the growing concern of off-target side effects due to OPP exposure has prompted the need for sensitive and economical detection methods. Here we set out to engineer a previously identified OPP responsive transcription factor, ChpR, from Sinorhizobium melilotii to respond to alternative OPPs and generate a repertoire of whole-cell biosensors for OPPs. The ChpR transcription factor and cognate promoter P chpA, have been shown to activate transcription in the presence of the OPP chlorpyrifos (CPF). Utilizing a GFP reporter regulated by ChpR in a whole-cell biosensor we found that the system responds significantly better to 3,5,6-trichloro-2-pyridinol (TCP), the main degradation product of CPF, compared to CPF itself. This biosensor was able to respond to TCP at 390 nM within 4 h compared to 50 µM of CPF in 7 h. The ChpR-P chpA , and the activating ligand TCP, were able to regulate expression of a kanamycin resistance/sucrose sensitivity (kan/sacB) selection/counterselection module suitable for high throughput mutagenesis screening studies. The ability to control both GFP and the kan/sacB module demonstrates the utility of this reporter for the detection of CPF affected areas. The ChpR-P chpA system serves as an additional positive regulator switch to add to the growing repertoire of controllers available within synthetic biology.

Probiotic strains from different origins have shown promise in recent decades for their health benefits, for example in promoting and regulating the immune system. The immunomodulatory potential of four Lactobacillus strains from animal and plant origins was evaluated in this paper based on their genomic information. Comparative genomic analysis was performed through genome alignment, average nucleotide identity (ANI) analysis and gene mining for putative immunomodulatory genes. The genomes of the four Lactobacillus strains show relative similarities in multiple regions, as observed in the genome alignment. However, ANI analysis showed that L. mucosae LM1 and L. fermentum SK152 are the most similar when considering their nucleotide sequences alone. Gene mining of putative immunomodulatory genes studied from L. plantarum WCFS1 yielded multiple results in the four potential probiotic strains, with L. plantarum SK151 showing the largest number of genes at around 74 hits, followed by L. johnsonii PF01 at 41 genes when adjusted for matches with at least 30 % identity. Looking at the immunomodulatory genes in each strain, L. plantarum SK151 and L. johnsonii PF01 may have wider activity, covering both immune activation and immune suppression, as compared to L. mucosae LM1 and L. fermentum SK152, which could be more effective in activating immune cells and the pro-inflammatory cascade rather than suppressing it. The similarities and differences between the four Lactobacillus species showed that there is no definitive trend based on the origin of isolation alone. Moreover, higher percentage identities between genomes do not directly correlate with higher similarities in potential activity, such as in immunomodulation. The immunomodulatory function of each of the four Lactobacillus strains should be observed and verified experimentally in the future, since some the activity of some genes may be strain-specific, which would not be identified through comparative genomics alone.

The Candida albicans high-affinity phosphate transporter Pho84 is required for normal Target of Rapamycin signaling, oxidative stress resistance and virulence of this fungal pathogen. It also contributes to C. albicans’ tolerance of two antifungal drug classes, polyenes and echinocandins. Echinocandins inhibit biosynthesis of a major cell wall component, beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including nucleotides like ATP and nucleotide sugars, were low in pho84 mutant compared to wild type cells recovering from phosphate starvation. Non-phosphoric precursors like nucleobases and nucleosides were elevated. Outer cell wall phosphomannan biosynthesis requires a nucleotide sugar,GDP-mannose. The nucleotide sugar UDP-glucose is the substrate of enzymes that synthesize two major structural cell wall polysaccharides, beta-1,3- and beta-1,6-glucan. Another nucleotide sugar, UDP-N-acetylglucosamine, is the substrate of chitin synthases which produce a stabilizing component of the intercellular septum and of lateral cell walls. Lack of Pho84 activity, and phosphate starvation, potentiated pharmacological or genetic perturbation of these enzymes. Our model is that low substrate concentrations of beta-D-glucan- and chitin synthases diminish enzymatic reaction rates and potentiate pharmacologic inhibitors to decrease the yield of their cell wall-stabilizing products. Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-D-glucans or chitin. Hence inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans.

Candida albicans colonises the gastro-intestinal (GI) tract of over 60% of the population. In severely ill or immune compromised patients, this fungus can escape the gut, disseminate through the body and cause systemic disease. Most research in the field has focused on defining traits that contribute directly to virulence; there are comparatively few studies which have addressed how C. albicans colonises and persists in the gut. Furthermore, such studies have typically been performed mouse models devoid of resident GI bacteria, completely neglecting the major impact of the local microbiota on GI colonisation. How, then, does C. albicans persist in the GI tract in the presence of the normal gut microbiota?

To address this question, a novel in vitro two-phase anaerobic fermentation system that simulates the human colon microenvironment has been developed. This “colon microcosm” supports the growth of human faecal microbiota in liquid anaerobic colon medium (phase 1) and C. albicans growth on agar plugs which are added to the medium to mimic the epithelial surface (phase 2). The impact of C. albicans upon the faecal microbiota is monitored by examining the planktonic phase (phase 1), whilst the effect of the microbiota on the growth of C. albicans is monitored after extracting C. albicans cells from the agar plugs (phase 2).

The results of assays carried out to validate the model will be presented, as will data from pilot studies which illustrate the potentially exploitable impact of the human GI microbiota from healthy individuals on C. albicans growth.

The relevance of C. glabrata as a human pathogen is linked with its poor susceptibility to azoles as well as its extreme genomic plasticity that allows the rapid acquisition of resistance. Extensive characterization of azole-resistant C. glabrata strains unveiled the central role of the transcriptional regulator CgPdr1 in the resistance phenotype, with many strains encoding hyperactive (or gain-of-function; GOF) CgPdr1 alleles. Large scale profiling of a collection of clinical C. glabrata isolates recovered in hospitals of the Lisbon area, in Portugal, led to the identification of 11 strains exhibiting resistance to fluconazole and voriconazole, while 2 were only resistant to fluconazole. Among these strains, 10 were found to encode alleles of the CgPDR1 gene harbouring multiple non-synonymous SNPs that were not found in the alleles encoded by susceptible strains, including K274Q, I392M and I803T not previously described as GOF mutations. The isolates encoding these alleles were found to over-express several CgPdr1 target genes including the azole efflux pump CgCDR1 sustaining the idea that these represent new gain-of-function CgPdr1 alleles. Only one of the identified azole-resistant strains was found to encode a CgPDR1 allele fully identical to the one encoded by susceptible strains. To better understand the resistance phenotype of this strain, its transcriptome was compared with the one of a susceptible strain and of strains encoding CgPdr1 GOF alleles. The results of this comparative transcriptomic analysis will be discussed shedding light into the different azole-resistance mechanisms evolved by C. glabrata, including those independent of CgPdr1 GOF strains.

The rise in drug resistance amongst pathogenic fungi, paired with the limited arsenal of antifungals available is an imminent threat to our medical system. To address this, we screened two distinct compound libraries to identify novel strategies to expand the antifungal armamentarium. The first collection wasthe RIKEN Natural Product Depository (NPDepo), which was screened for antifungal activity against four major human fungal pathogens: Candida albicans, Candida glabrata, Candida auris, and Cryptococcus neoformans. Through a prioritization pipeline, one compound, NPD6433, emerged as having broad-spectrum antifungal activity and minimal mammalian cytotoxicity. Chemical-genetic and biochemical assays demonstrated that NPD6433 inhibits the essential fungal enzyme fatty acid synthase 1 (Fas1). Treatment with NPD6433 inhibited various virulence traits in C. neoformans and C. auris, and rescued mammalian cell growth in a co-culture model with C. auris. The second compound library screened was adiversity-oriented collectionfrom Boston University. This chemical screen was focused on identifying novel molecules that enhance the activity of the widely deployed antifungal, fluconazole, against C. auris. Through this endeavour, we discovered a potent compound that enhanced fluconazole efficacy against C. auris through increasing azole intracellular accumulation. This activity was dependent on expression of the multidrug transporter geneCDR1, suggesting that this compound targets efflux mechanisms. Furthermore, this molecule significantly reduced fungal burden alone and in combination with fluconazole in a murine model of C. auris disseminated infection. Overall, this work identifies novel compounds with bioactivity against fungal pathogens, revealing important biology, and paving the way for the critical development of therapeutic strategies.

Alternative gene splicing (AS) is a process by which a single gene can give rise to different protein isoforms, generating proteome diversity. Despite recent advances in our understanding of AS in basic cellular processes, the role of AS in drug resistance and fungal pathogenesis is poorly understood. In Candida albicans, approximately 6% of the genes contain introns. Considering this low and random distribution of introns, we focused our study on alternative splicing (AS) and its impact on the development of drug resistance, an area largely unexplored in this yeast. We performed comparative RNA sequencing of sequential isogenic azole sensitive and resistant isolates of C. albicans. The analysis revealed differential expression of splice junctions/isoforms in 14 genes, between the drug sensitive and resistant isolates. Furthermore, C. albicans WT cells exposed to antifungal drugs, heat stress or metal deficiency also showed differential expression of isoforms for the genes undergoing AS. In this study we present data on the effect of AS on the function of SOD3. The C. albicans SOD3 has a single intron and is important for the removal of superoxide radicals. The overexpression of the two isoforms of SOD3 in its null background highlighted importance of spliced isoform in complementing the susceptibility to menadione. However, the two isoforms did not differ in rescuing the susceptibility of sod3Δ/Δto Amphotericin B. Collectively, these data suggest that AS may be a novel mechanism in C. albicans for stress adaptation and overcoming drug resistance.

Candida albicans is a commensal yeast of the human gut, which is tolerated by the immune system, but has the potential to become an opportunistic pathogen. One way in which C. albicans achieves this duality is through concealing, or exposing cell wall pathogen-associated molecular patterns (PAMPs) in response to host derived environment cues (pH, hypoxia, lactate). This cell wall remodelling allows C. albicans to evade or hyperactivate the host’s innate immune responses leading to disease. Previously, we identified that adaptation of C. albicans to acidic environments, conditions encountered during colonisation of the female reproductive tract, induce significant cell wall remodelling resulting in the exposure of two key fungal PAMPs (glucan and chitin). Here we report that this pH-dependent cell wall remodelling is time dependent with the initial change in pH driving cell wall unmasking, which is then remasked at later time points. Remasking ofglucan was mediated via the cell density dependent fungal quorum sensing molecule farnesol, while chitin remasking was mediated via a small, heat-stable, non-proteinaceous secreted molecule(s). Transcript profiling identified a core set of 42 genes significantly regulated by pH over time, and identified the transcription factor Efg1 as a regulator of chitin exposure through regulation of CHT2. This dynamic cell wall remodelling influenced innate immune recognition of C. albicans, suggesting that during infection C. albicans can manipulate the host innate immune responses.

Candida glabrata-caused candidiasis is growing but treatments remain limited by paucity of drug targets, intrinsic azole resistance and increasing resistance to other drug classes. Drug resistance is one of numerous virulence traits regulated by the chaperone, heat shock protein 90 (Hsp90) in Candida albicans via its interactions with 5% of the genome. Hsp90 also regulates key drug resistance mechanisms in C. glabrata, but little else was known about Hsp90 in this organism. Therefore, CgHsp90 interactions were elucidated by genetic and proteomic methods.

A genetic network was produced by a chemical-genetic, synthetic-sick screen on a gene-deletion library covering 16% of the genome; whilst quantitative proteomics was undertaken by tandem mass tagging on wild-type cells. Both experiments were undertaken at 30°C, 37°C and 39°C and Hsp90 was perturbed using sub-lethal concentrations of Hsp90 inhibitor. Efforts to identify Hsp90 interactors at these host-infection associated temperatures produced a genetic network of 68 genes and a protein network of 2298 proteins. Of these, 4 genes and 261 proteins interacted with Hsp90 at all three temperatures, indicating a core Hsp90 interaction network. Intriguingly, both networks had enrichment for the “antibiotic biosynthesis” GO term. Two genes, BCY1 and MCM16, were found to interact with Hsp90 at multiple temperatures in both networks. These data indicate the divergence of Hsp90 networks between C. glabrata and its close relatives and offer important targets for further research. Presented here is the first multi-omic interaction network in C. glabrata, focused on the virulence and drug resistance regulator, Hsp90.

The primary recognition event between a fungal pathogen and the immune system normally involves the engagement of a pattern recognition receptor with specific components of the cell wall. However, the cell wall is a complex three dimensional structure whose composition changes rapidly in accordance with environmental stimuli. Therefore it is important to know what is the precise nature of the primary recognition event, how many events occur to activate the immune response and how these recognition events are affected by changes in cell wall architecture, cellular morphogenesis and physiological adaptation of the pathogen to specific niches in the human body. We address this fundamental question using four soluble immune C-Type lectin receptor-probes which recognize specific mannans and β-1,3 glucan in the cell wall. We use these C-type lectin probes to demonstrate that mannan epitopes are differentially distributed in the inner and outer layers of fungal cell wall in a clustered or diffuse manner. Immune reactivity of fungal cell surfaces did not correlate with relatedness of different fungal species, and mannan-detecting receptor-probes discriminated between cell surface mannans generated by the same fungus growing under different conditions. These studies demonstrate that mannan-epitopes within fungal cell walls are differentially distributed and dynamically expressed as the fungus adapted to microenvironments that would be encountered in vivo.

The Multidrug resistance-1 protein (Mdr1p) of Candida albicans is a crucial drug/H+ antiporter within the Major Facilitator Superfamily of proteins involved in the efflux of a broad spectrum of structurally diverse xenobiotic compounds. As a member of the DHA1 subfamily, Mdr1p consists of 12 transmembrane helices (TMHs), divided equally into two Transmembrane Domains (TMDs). How the pseudo-symmetrically positioned TMHs, and the TMDs they compose, communicate with each other remains poorly characterized. In that direction, the recovery of spontaneous chromosomal mutants that negatively affect the primary mutant phenotype can provide essential inter-domain communication insights. For this purpose, in the current study we have performed a suppressor screen for a critically transport deficient mutant G230A, located within TMH-4 of Mdr1p, predicted towards the intracellular space. The recovered suppressor (P528H), that restores the transport capacity of this initially drug susceptible mutant, map to TMH-12, very close to the extracellular space. Since the mutant and suppressor sites occupy the N-domain and C-domain, respectively, and locate at a pseudo-symmetrical position, these results hint to a novel pattern of crosstalk. Additionally, the recovered suppressor mutation restores wild type phenotypes for all tested xenobiotic substrates except cycloheximide, thus implying substrate selectivity. Furthermore, the molecular modeling and docking results suggest a novel compensatory mechanism which is independent of drug binding. Altogether, the present study is a first attempt to gain insights into the transport mechanism of drug/H+ antiporter using the suppressor genetics approach.

Candida auris is globally recognized as an opportunistic fungal pathogen of high concern, due to its extensive multidrug-resistance (MDR). Still, molecular mechanisms of MDR are largely unexplored. This is the first account of genome wide evolution of MDR in C. auris obtained through serial in vitro exposure to azoles, polyenes and echinocandins. We show the stepwise accumulation of multiple novel mutations in genes known and unknown in antifungal drug resistance, albeit almost all new for C. auris. Echinocandin resistance was accompanied by a codon deletion in FKS1hot spot 1 and a substitution in FKS1 ‘novel’ hot spot 3. Mutations in ERG3 and CIS2 further increased the echinocandin MIC. Decreased azole susceptibility was linked to a mutation in transcription factor TAC1b and overexpression of the drug efflux pump Cdr1; a segmental duplication of chromosome 1 containing ERG11; and a whole chromosome 5 duplication, which contains TAC1b. The latter was associated with increased expression of ERG11, TAC1band CDR2, but not CDR1. The simultaneous emergence of nonsense mutations in ERG3 and ERG11 was shown to decrease amphotericin B susceptibility, accompanied with fluconazole cross resistance. A mutation in MEC3, a gene mainly known for its role in DNA damage homeostasis, further increased the polyene MIC. Overall, this study shows the alarming potential and diversity for MDR development in C. auris, even in a clade until now not associated with MDR (clade II),hereby stressing its clinical importance and the urge for future research.