Spontaneous Suppressors against Debilitating Transmembrane Mutants of CaMdr1 Disclose Novel Interdomain Communication via Signature Motifs of the Major Facilitator Superfamily.

The Major Facilitator Superfamily (MFS) drug:H+ antiporter CaMdr1, from Candida albicans, is responsible for the efflux of structurally diverse antifungals. MFS members share a common fold of 12−14 transmembrane helices (TMHs) forming two N- and C-domains. Each domain is arranged in a pseudo-symmetric fold of two tandems of 3-TMHs that alternatively expose the drug-binding site towards the inside or the outside of the yeast to promote drug binding and release. MFS proteins show great diversity in primary structure and few conserved signature motifs, each thought to have a common function in the superfamily, although not yet clearly established. Here, we provide new information on these motifs by having screened a library of 64 drug transport-deficient mutants and their corresponding suppressors spontaneously addressing the deficiency. We found that five strains recovered the drug-resistance capacity by expressing CaMdr1 with a secondary mutation. The pairs of debilitating/rescuing residues are distributed either in the same TMH (T127ATMH1- > G140DTMH1) or 3-TMHs repeat (F216ATMH4- > G260ATMH5), at the hinge of 3-TMHs repeats tandems (R184ATMH3- > D235HTMH4, L480ATMH10- > A435TTMH9), and finally between the N- and C-domains (G230ATMH4- > P528HTMH12). Remarkably, most of these mutants belong to the different signature motifs, highlighting a mechanistic role and interplay thought to be conserved among MFS proteins. Results also point to the specific role of TMH11 in the interplay between the N- and C-domains in the inward- to outward-open conformational transition.

Fusobacterium nucleatum is associated with inflammation and poor survival in early-stage HPV-negative tongue cancer.

Persistent pathogen infection is a known cause of malignancy, although with sparse systematic evaluation across tumor types. We present a comprehensive landscape of 1060 infectious pathogens across 239 whole exomes and 1168 transcriptomes of breast, lung, gallbladder, cervical, colorectal, and head and neck tumors. We identify known cancer-associated pathogens consistent with the literature. In addition, we identify a significant prevalence of Fusobacterium in head and neck tumors, comparable to colorectal tumors. The Fusobacterium-high subgroup of head and neck tumors occurs mutually exclusive to human papillomavirus, and is characterized by overexpression of miRNAs associated with inflammation, elevated innate immune cell fraction and nodal metastases. We validate the association of Fusobacterium with the inflammatory markers IL1B, IL6 and IL8, miRNAs hsa-mir-451a, hsa-mir-675 and hsa-mir-486-1, and MMP10 in the tongue tumor samples. A higher burden of Fusobacterium is also associated with poor survival, nodal metastases and extracapsular spread in tongue tumors defining a distinct subgroup of head and neck cancer.

A one-step, one-tube real-time RT-PCR based assay with an automated analysis for detection of SARS-CoV-2.

Early diagnosis of SARS-CoV-2 infected patients is essential to control the dynamics of the COVID-19 pandemic. We develop a rapid and accurate one-step multiplex TaqMan probe-based real-time RT-PCR assay, along with a computational tool to systematically analyse the data. Our assay could detect to a limit of 15 copies of SARS-CoV-2 transcripts-based on experiments performed by spiking total human RNA with in vitro synthesized viral transcripts. The assay was evaluated by performing 184 validations for the SARS-CoV-2 Nucleocapsid gene and human RNase P as an internal control reference gene with dilutions ranging from 1-100 ng for human RNA on a cohort of 26 clinical samples. 5 of 26 patients were confirmed to be infected with SARS-CoV-2, while 21 tested negative, consistent with the standards. The accuracy of the assay was found to be 100% sensitive and 100% specific based on the 26 clinical samples that need to be further verified using a large number of clinical samples. In summary, we present a rapid, easy to implement real-time PCR based assay with automated analysis using a novel COVID qPCR Analyzer tool with graphical user interface (GUI) to analyze the raw qRT-PCR data in an unbiased manner at a cost of under $3 per reaction and turnaround time of less than 2h, to enable in-house SARS-CoV-2 testing across laboratories.

Mitochondrial glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase abrogate p53 induced apoptosis in a yeast model: Possible implications for apoptosis resistance in cancer cells.

BACKGROUND: Escape from apoptosis is an important hallmark of tumor progression and drug resistance in cancer cells. It is well demonstrated that over-expression of human wtp53 in Saccharomyces cerevisiae induces apoptosis by directly targeting the mitochondria. In this study, we showed that how S.cerevisiae escaped from p53 induced apoptosis in the presence of a fermentable carbon source (sucrose), but not on non-fermentable carbon source (glycerol). METHODS: Mitochondrial fractions from yeast cultures grown in the presence of sucrose or glycerol with and without p53 expression were fractionated and analyzed by LC-MS/MS. Differentially expressed proteins were studied and detailed biochemical analysis for selected proteins was performed.The effect of mitochondrial HXK-2 over-expression induced by p53 in sucrose grown cells on cell survival was evaluated using gene deletion/tagging, co-localisation and mitochondrial ROS detection. RESULTS: We observe that mitochondria isolated from p53 over-expressing cells accumulate Pentose phosphate Pathway (PPP) enzymes including glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) which led to enhanced mitochondrial NADPH production only when cells are cultured in sucrose but not glycerol. In contrast, mitochondria isolated from Δhxk2 p53 over-expressing cells grown in sucrose did not accumulate G6PDH and 6PGDH and resulted in defective growth. CONCLUSIONS: Enhanced association of HXK2 with the mitochondria with the concomitant accumulation of G6PDG and 6PGDH results in increased NADPH that scavenges ROS and provides resistance to apoptosis. GENERAL SIGNIFICANCE: Given the extensive similarity of aerobic glycolysis between humans and yeast, the phenomena described here could as well be responsible for the escape of apoptosis in cancer cells.

W1038 near D-loop of NBD2 is a focal point for inter-domain communication in multidrug transporter Cdr1 of Candida albicans.

Candida drug resistance 1 (Cdr1), a PDR subfamily ABC transporter mediates efflux of xenobiotics in Candida albicans. It is one of the prime factors contributing to multidrug resistance in the fungal pathogen. One hallmark of this transporter is its asymmetric nature, characterized by peculiar alterations in its nucleotide binding domains. As a consequence, there exists only one canonical ATP-binding site while the other is atypical. Here, we report suppressor analysis on the drug-susceptible transmembrane domain mutant V532D that identified the suppressor mutation W1038S, close to the D-loop of the non-catalytic ATP-binding site. Introduction of the W1038S mutation in the background of V532D mutant conferred resistance for most of the substrates to the latter. Such restoration is accompanied by a severe reduction of ATPase activity, of about 85%, while that of the V532D mutant is half-reduced. Conversely, alanine substitution of the highly conserved aspartate D1033A in that D-loop rendered cells selectively hyper-susceptible to miconazole without an impact on steady-state ATPase activity, suggesting altogether that ATP hydrolysis may not hold the key to restoration mechanism. Analysis of the ABCG5/ABCG8-based 3D-model of Cdr1p suggested that the W1038S substitution leads to the loss of hydrophobic interactions and H-bond with residues of the neighbor NBD1, in the non-catalytic ATP-binding site area. The compensatory effect within TMDs accounting for transport restoration in the V532D-W1038S variant may, therefore, be mainly due to an increase in NBDs mobility at the non-catalytic interface.

Molecular Basis of Substrate Polyspecificity of the Candida albicans Mdr1p Multidrug/H+ Antiporter.

The molecular basis of polyspecificity of Mdr1p, a major drug/H+ antiporter of Candida albicans, is not elucidated. We have probed the nature of the drug-binding pocket by performing systematic mutagenesis of the 12 transmembrane segments. Replacement of the 252 amino acid residues with alanine or glycine yielded 2/3 neutral mutations while 1/3 led to the complete or selective loss of resistance to drugs or substrates transported by the pump. Using the GlpT-based 3D-model of Mdr1p, we roughly categorized these critical residues depending on their type and localization, 1°/ main structural impact ("S" group), 2°/ exposure to the lipid interface ("L" group), 3°/ buried but not facing the main central pocket, inferred as critical for the overall H+/drug antiport mechanism ("M" group) and finally 4°/ buried and facing the main central pocket ("B" group). Among "B" category, 13 residues were essential for the large majority of drugs/substrates, while 5 residues were much substrate-specific, suggesting a role in governing polyspecificity (P group). 3D superposition of the substrate-specific MFS Glut1 and XylE with the MDR substrate-polyspecific MdfA and Mdr1p revealed that the B group forms a common substrate interaction core while the P group is only found in the 2 MDR MFS transporters, distributed into 3 areas around the B core. This specific pattern has let us to propose that the structural basis for polyspecificity of MDR MFS transporters is the extended capacity brought by residues located at the periphery of a binding core to accomodate compounds differing in size and type.

pHluorin enables insights into the transport mechanism of antiporter Mdr1: R215 is critical for drug/H+ antiport.

Multidrug resistance 1 (MDR1) is a member of the major facilitator superfamily that contributes to MDR of Candida albicans This antiporter belongs to the drug/H(+) antiporter 1 family, pairing the downhill gradient of protons to drug extrusion. Hence, drug efflux from cytosol to extracellular space and the parallel import of H(+) towards cytosol are inextricably linked processes. For monitoring the drug/H(+) antiporter activity of Mdr1p, we developed a new system, exploiting a GFP variant pHluorin, which changes its fluorescence properties with pH. This enabled us to measure the cytosolic pH correlated to drug efflux. Since protonation of charged residues is a key step in proton movement, we explored the role of all charged residues of the 12 transmembrane segments (TMSs) of Mdr1p in drug/H(+) transport by mutational analysis. This revealed that the conserved residue R(215), positioned close to the C-terminal end of TMS-4, is critical for drug/H(+) antiport, allowing protonation over a range of pH, in contrast with its H(215) or K(215) variants that failed to transport drugs at basic pH. Mutation of other residues of TMS-4 highlights the role of this TMS in drug transport, as confirmed by in silico modelling of Mdr1p and docking of drugs. The model points to the importance of R(215) in proton transport, suggesting that it may adopt two main conformations, one oriented towards the extracellular face and the other towards the centre of Mdr1p. Together, our results not only establish a new system for monitoring drug/H(+) transport, but also unveil a positively charged residue critical to Mdr1p function.