Deletion of the Candida albicans TLO gene family using CRISPR-Cas9 mutagenesis allows characterisation of functional differences in α-, ß- and γ- TLO gene function.

The Candida albicans genome contains between ten and fifteen distinct TLO genes that all encode a Med2 subunit of Mediator. In order to investigate the biological role of Med2/Tlo in C. albicans we deleted all fourteen TLO genes using CRISPR-Cas9 mutagenesis. ChIP-seq analysis showed that RNAP II localized to 55% fewer genes in the tloΔ mutant strain compared to the parent, while RNA-seq analysis showed that the tloΔ mutant exhibited differential expression of genes required for carbohydrate metabolism, stress responses, white-opaque switching and filamentous growth. Consequently, the tloΔ mutant grows poorly in glucose- and galactose-containing media, is unable to grow as true hyphae, is more sensitive to oxidative stress and is less virulent in the wax worm infection model. Reintegration of genes representative of the α-, ß- and γ-TLO clades resulted in the complementation of the mutant phenotypes, but to different degrees. TLOα1 could restore phenotypes and gene expression patterns similar to wild-type and was the strongest activator of glycolytic and Tye7-regulated gene expression. In contrast, the two γ-TLO genes examined (i.e., TLOγ5 and TLOγ11) had a far lower impact on complementing phenotypic and transcriptomic changes. Uniquely, expression of TLOß2 in the tloΔ mutant stimulated filamentous growth in YEPD medium and this phenotype was enhanced when Tloß2 expression was increased to levels far in excess of Med3. In contrast, expression of reintegrated TLO genes in a tloΔ/med3Δ double mutant background failed to restore any of the phenotypes tested, suggesting that complementation of these Tlo-regulated processes requires a functional Mediator tail module. Together, these data confirm the importance of Med2/Tlo in a wide range of C. albicans cellular activities and demonstrate functional diversity within the gene family which may contribute to the success of this yeast as a coloniser and pathogen of humans.

Investigation of genomic mutations and their association with phenotypic resistance to new and repurposed drugs in Mycobacterium tuberculosis complex clinical isolates.

BACKGROUND: WGS has the potential to detect resistance-associated mutations and guide treatment of MDR TB. However, the knowledge base to confidently interpret mutations associated with the new and repurposed drugs is sparse, and phenotypic drug susceptibility testing is required to detect resistance. METHODS: We screened 900 Mycobacterium tuberculosis complex genomes from Ireland, a low TB incidence country, for mutations in 13 candidate genes and assessed their association with phenotypic resistance to bedaquiline, clofazimine, linezolid, delamanid and pretomanid. RESULTS: We identified a large diversity of mutations in the candidate genes of 195 clinical isolates, with very few isolates associated with phenotypic resistance to bedaquiline (n = 4), delamanid (n = 4) and pretomanid (n = 2). We identified bedaquiline resistance among two drug-susceptible TB isolates that harboured mutations in Rv0678. Bedaquiline resistance was also identified in two MDR-TB isolates harbouring Met146Thr in Rv0678, which dated back to 2007, prior to the introduction of bedaquiline. High-level delamanid resistance was observed in two isolates with deletions in ddn, which were also resistant to pretomanid. Delamanid resistance was detected in two further isolates that harboured mutations in fbiA, but did not show cross-resistance to pretomanid. All isolates were susceptible to linezolid and clofazimine, and no mutations found were associated with resistance. CONCLUSIONS: More studies that correlate genotypic and phenotypic drug susceptibility data are needed to increase the knowledge base of mutations associated with resistance, in particular for pretomanid. Overall, this study contributes to the development of future mutation catalogues for M. tuberculosis complex isolates.

Stress- and metabolic responses of Candida albicans require Tor1 kinase N-terminal HEAT repeats.

Whether to commit limited cellular resources toward growth and proliferation, or toward survival and stress responses, is an essential determination made by Target of Rapamycin Complex 1 (TORC1) for a eukaryotic cell in response to favorable or adverse conditions. Loss of TORC1 function is lethal. The TORC1 inhibitor rapamycin that targets the highly conserved Tor kinase domain kills fungal pathogens like Candida albicans, but is also severely toxic to human cells. The least conserved region of fungal and human Tor kinases are the N-terminal HEAT domains. We examined the role of the 8 most N-terminal HEAT repeats of C. albicans Tor1. We compared nutritional- and stress responses of cells that express a message for N-terminally truncated Tor1 from repressible tetO, with cells expressing wild type TOR1 from tetO or from the native promoter. Some but not all stress responses were significantly impaired by loss of Tor1 N-terminal HEAT repeats, including those to oxidative-, cell wall-, and heat stress; in contrast, plasma membrane stress and antifungal agents that disrupt plasma membrane function were tolerated by cells lacking this Tor1 region. Translation was inappropriately upregulated during oxidative stress in cells lacking N-terminal Tor1 HEAT repeats despite simultaneously elevated Gcn2 activity, while activation of the oxidative stress response MAP kinase Hog1 was weak. Conversely, these cells were unable to take advantage of favorable nutritional conditions by accelerating their growth. Consuming oxygen more slowly than cells containing wild type TOR1 alleles during growth in glucose, cells lacking N-terminal Tor1 HEAT repeats additionally were incapable of utilizing non-fermentable carbon sources. They were also hypersensitive to inhibitors of specific complexes within the respiratory electron transport chain, suggesting that inefficient ATP generation and a resulting dearth of nucleotide sugar building blocks for cell wall polysaccharides causes cell wall integrity defects in these mutants. Genome-wide expression analysis of cells lacking N-terminal HEAT repeats showed dysregulation of carbon metabolism, cell wall biosynthetic enzymes, translational machinery biosynthesis, oxidative stress responses, and hyphal- as well as white-opaque cell type-associated genes. Targeting fungal-specific Tor1 N-terminal HEAT repeats with small molecules might selectively abrogate fungal viability, especially when during infection multiple stresses are imposed by the host immune system.

Draft Genome Sequences of Clinical Respiratory Isolates of Mycobacterium goodii Recovered in Ireland.

Here, we describe the draft genomes of five Mycobacterium goodii isolates that were recovered from respiratory clinical specimens in Ireland. Currently, one complete genome and one draft genome exist publicly for M. goodii.

False detection of rifampicin resistance using Xpert® MTB/RIF Ultra assay due to an A451V mutation in Mycobacterium tuberculosis.

BACKGROUND: In a 12 month period, three Irish-born adult cases with pulmonary TB were initially diagnosed by Xpert® MTB/RIF Ultra assay, which detected a rifampicin resistance-conferring mutation prompting treatment as potential MDR cases. METHODS: Further laboratory investigations on the cultured isolates included GenoType MTBDRplus assay, phenotypic drug susceptibility tests using the BD BACTEC MGIT culture system and MIC broth microdilution tests. Sequencing of the rpoB gene was performed using Sanger sequencing and WGS. RESULTS: Phenotypic drug susceptibility tests determined the isolates to be rifampicin susceptible. Molecular investigations identified an A451V (codon 532) mutation in the Mycobacterium tuberculosis rpoB gene that has not previously been found to cause rifampicin resistance. Genome sequencing revealed that the three isolates' genomes differed by ≤5 SNPs, indicating a high likelihood of recent transmission events. Furthermore, a cluster of six related M. tuberculosis isolates from our in-house typing database showed four were highly related; all were rifampicin susceptible and lacked this mutation. CONCLUSIONS: False detection of rifampicin resistance, albeit rare, should be considered possible with Xpert® MTB/RIF Ultra assay, particularly in low TB incidence settings. Confirmatory sequencing methods should be performed to prevent the unnecessary use of second-line anti-tuberculous drugs.

Overcoming the Challenges of Pyrazinamide Susceptibility Testing in Clinical Mycobacterium tuberculosis Isolates.

Pyrazinamide (PZA) is one of the first-line agents used for the treatment of tuberculosis. However, current phenotypic PZA susceptibility testing in the Bactec MGIT 960 system is unreliable, and false resistance is well documented. Rapid identification of resistance-associated mutations can confirm the phenotypic result. This study aimed to investigate the use of genotypic methods in combination with phenotypic susceptibility testing for confirmation of PZA-resistant Mycobacterium tuberculosis isolates. Sanger sequencing and/or whole-genome sequencing were performed to detect mutations in pncA, rpsA, panD, and clpC1. Isolates were screened for heteroresistance, and PZA susceptibility testing was performed using the Bactec MGIT 960 system using a reduced inoculum to investigate false resistance. Overall, 40 phenotypically PZA-resistant isolates were identified. Of these, PZA resistance was confirmed in 22/40 (55%) isolates by detecting mutations in the pncA, rpsA, and panD genes. Of the 40 isolates, 16 (40%) were found to be susceptible using the reduced inoculum method (i.e., false resistance). No mutations were detected in two PZA-resistant isolates. False resistance was observed in isolates with MICs close to the critical concentration. In particular, East African Indian strains (lineage 1) appeared to have an elevated MIC that is close to the critical concentration. While this study illustrates the complexity and challenges associated with PZA susceptibility testing of M. tuberculosis, we conclude that a combination of genotypic and phenotypic drug susceptibility testing methods is required for accurate detection of PZA resistance.

Genomic Analysis of Mycobacterium abscessus Complex Isolates Collected in Ireland between 2006 and 2017.

Members of the Mycobacterium abscessus complex (MABC) are multidrug-resistant nontuberculous mycobacteria and cause opportunistic pulmonary infections in individuals with cystic fibrosis (CF). In this study, genomic analysis of MABC isolates was performed to gain greater insights into the epidemiology of circulating strains in Ireland. Whole-genome sequencing (WGS) was performed on 70 MABC isolates that had been referred to the Irish Mycobacteria Reference Laboratory between 2006 and 2017 across nine Irish health care centers. The MABC isolates studied comprised 52 isolates from 27 CF patients and 18 isolates from 10 non-CF patients. WGS identified 57 (81.4%) as M. abscessus subsp. abscessus, 10 (14.3%) as M. abscessus subsp. massiliense, and 3 (4.3%) as M. abscessus subsp. bolletii Forty-nine (94%) isolates from 25 CF patients were identified as M. abscessus subsp. abscessus, whereas 3 (6%) isolates from 2 CF patients were identified as M. abscessus subsp. massiliense Among the isolates from non-CF patients, 44% (8/18) were identified as M. abscessus subsp. abscessus, 39% (7/18) were identified as M. abscessus subsp. massiliense, and 17% (3/18) were identified as M. abscessus subsp. bolletii WGS detected two clusters of closely related M. abscessus subsp. abscessus isolates that included isolates from different CF centers. There was a greater genomic diversity of MABC isolates among the isolates from non-CF patients than among the isolates from CF patients. Although WGS failed to show direct evidence of patient-to-patient transmission among CF patients, there was a predominance of two different strains of M. abscessus subsp. abscessus Furthermore, some MABC isolates were closely related to global strains, suggesting their international spread. Future prospective real-time epidemiological and clinical data along with contemporary MABC sequence analysis may elucidate the sources and routes of transmission among patients infected with MABC.

First description of arginine catabolic mobile element (ACME) type VI harboring the kdp operon only in Staphylococcus epidermidis using short and long read whole genome sequencing: Further evidence of ACME diversity.

The arginine catabolic mobile element (ACME) was first described in methicillin-resistant Staphylococcus aureus and is considered to enhance transmission, persistence and survival. Subsequently ACMEs were shown to be more prevalent in the coagulase-negative Staphylococcus epidermidis. Previously, ACME types were distinguished by characteristic combinations of the arc and opp3 operons [I (arc+, opp3+), II (arc+, opp3-) and III (arc-, opp3+)] encoding an arginine deaminase pathway and oligopeptide permease transporter, respectively. Recently two novel ACME types harboring the potassium transporter-encoding operon kdp were described in oral S. epidermidis isolates [IV (arc+, opp3-, kdp+), and V (arc+, opp3+, kdp+)]. This study investigated two independent oral S. epidermidis isolates that yielded amplimers with kdp-directed primers only when subjected to ACME typing PCRs. Hybrid assemblies based on Illumina MiSeq short-read and Oxford Nanopore MinION long-read whole genome sequences revealed that both isolates harbored a sixth, novel ACME type (VI) integrated into orfX. Both ACME VIs lacked the arc and opp3 operons, harbored the kdp operon adjacent to other commonly ACME-associated genes including speG, hsd, sdr, and rep, but the structural organization of the adjacent regions were distinct. These ACMEs were flanked by different direct repeat sequences and the ACME VI-positive isolates belonged to unrelated genetic clusters. Overall these findings are indicative of independent evolution. The identification of ACME type VI further illustrates the diversity of ACME elements in S. epidermidis. The presence of ACMEs harboring kdp may confer a selective advantage on oral S. epidermidis in a potassium-rich environment such as found in dental plaque.

Expansion of the TLO gene family enhances the virulence of Candida species.

The TLO genes are a family of subtelomeric ORFs in the fungal pathogens Candida albicans and C. dubliniensis encoding a subunit of the Mediator complex homologous to Med2. The more virulent pathogen C. albicans has 15 copies of the gene whereas the less pathogenic species C. dubliniensis has only two. To investigate if expansion of the TLO repertoire in C. dubliniensis has an effect on phenotype and virulence we expressed three representative C. albicans TLO genes (TLOß2, TLOγ11 and TLOα12) in a wild type C. dubliniensis background, under the control of either their native or the ACT1 promoter. Expression of TLOß2 resulted in a hyperfilamentous phenotype, while overexpression of TLOγ11 and TLOα12 resulted in enhanced resistance to oxidative stress. Expression of all three TLO genes from the ACT1 promoter resulted in increased virulence in the Galleria infection model. In order to further investigate if individual TLO genes exhibit differences in function we expressed six representative C. albicans TLO genes in a C. dubliniensis Δtlo1/Δtlo2 double mutant. Differences were observed in the ability of the expressed CaTLOs to complement the various phenotypes of the mutant. All TLO genes with the exception of TLOγ7 could restore filamentation, however only TLOα9, γ11 and α12 could restore chlamydospore formation. Differences in the ability of CaTLO genes to restore growth in the presence of H2O2, calcofluor white, Congo red and at 42°C were observed. Only TLOα3 restored wild-type levels of virulence in the Galleria infection model. These data show that expansion of the TLO gene family in C. dubliniensis results in gain of function and that there is functional diversity amongst members of the gene family. We propose that this expansion of the TLO family contributes to the success of C. albicans as a commensal and opportunistic pathogen.

The Candida albicans TOR-Activating GTPases Gtr1 and Rhb1 Coregulate Starvation Responses and Biofilm Formation.

Target of rapamycin complex 1 (TORC1) is an essential regulator of metabolism in eukaryotic cells and in the fungal pathogen Candida albicans regulates morphogenesis and nitrogen acquisition. Gtr1 encodes a highly conserved GTPase that in Saccharomyces cerevisiae regulates nitrogen sensing and TORC1 activation. Here, we characterize the role of C. albicans GTR1 in TORC1 activation and compare it with the previously characterized GTPase Rhb1. A homozygous gtr1/gtr1 mutant exhibited impaired TORC1-mediated phosphorylation of ribosomal protein S6 and increased susceptibility to rapamycin. Overexpression of GTR1 impaired nitrogen starvation-induced filamentous growth, MEP2 expression, and growth in bovine serum albumin as the sole nitrogen source. Both GTR1 and RHB1 were shown to regulate genes involved in ribosome biogenesis, amino acid biosynthesis, and expression of biofilm growth-induced genes. The rhb1/rhb1 mutant exhibited a different pattern of expression of Sko1-regulated genes and increased susceptibility to Congo red and calcofluor white. The homozygous gtr1/gtr1 mutant exhibited enhanced flocculation phenotypes and, similar to the rhb1/rhb1 mutant, exhibited enhanced biofilm formation on plastic surfaces. In summary, Gtr1 and Rhb1 link nutrient sensing and biofilm formation and this connectivity may have evolved to enhance the competitiveness of C. albicans in niches where there is intense competition with other microbes for space and nutrients. IMPORTANCECandida albicans is the major fungal pathogen of humans and is responsible for a wide range of infections, including life-threatening systemic infections in susceptible hosts. Target of rapamycin complex 1 (TORC1) is an essential regulator of metabolism in this fungus, and components of this complex are under increased investigation as targets for new antifungal drugs. The present study characterized the role of GTR1, encoding a putative GTPase, in TORC1 activation. This study shows that GTR1 encodes a protein required for activation of TORC1 activity in response to amino acids and regulation of nitrogen starvation responses. GTR1 mutants show increased cell-cell adhesion and biofilm formation and increased expression of genes involved in these processes. This study demonstrates that starvation responses and biofilm formation are coregulated by GTR1 and suggests that these responses are linked to compete with the microbiome for space and nutrients.

Phosphate is the third nutrient monitored by TOR in Candida albicans and provides a target for fungal-specific indirect TOR inhibition.

The Target of Rapamycin (TOR) pathway regulates morphogenesis and responses to host cells in the fungal pathogen Candida albicans Eukaryotic Target of Rapamycin complex 1 (TORC1) induces growth and proliferation in response to nitrogen and carbon source availability. Our unbiased genetic approach seeking unknown components of TORC1 signaling in C. albicans revealed that the phosphate transporter Pho84 is required for normal TORC1 activity. We found that mutants in PHO84 are hypersensitive to rapamycin and in response to phosphate feeding, generate less phosphorylated ribosomal protein S6 (P-S6) than the WT. The small GTPase Gtr1, a component of the TORC1-activating EGO complex, links Pho84 to TORC1. Mutants in Gtr1 but not in another TORC1-activating GTPase, Rhb1, are defective in the P-S6 response to phosphate. Overexpression of Gtr1 and a constitutively active Gtr1Q67L mutant suppresses TORC1-related defects. In Saccharomyces cerevisiae pho84 mutants, constitutively active Gtr1 suppresses a TORC1 signaling defect but does not rescue rapamycin hypersensitivity. Hence, connections from phosphate homeostasis (PHO) to TORC1 may differ between C. albicans and S. cerevisiae The converse direction of signaling from TORC1 to the PHO regulon previously observed in S. cerevisiae was genetically shown in C. albicans using conditional TOR1 alleles. A small molecule inhibitor of Pho84, a Food and Drug Administration-approved drug, inhibits TORC1 signaling and potentiates the activity of the antifungals amphotericin B and micafungin. Anabolic TORC1-dependent processes require significant amounts of phosphate. Our study shows that phosphate availability is monitored and also controlled by TORC1 and that TORC1 can be indirectly targeted by inhibiting Pho84.