Mycobacterium tuberculosis (Mtb) strains are classified into different phylogenetic lineages (L), three of which (L2/L3/L4) emerged from a common progenitor after the loss of the MmpS6/MmpL6-encoding Mtb-specific deletion 1 region (TbD1). These TbD1-deleted “modern” lineages are responsible for globally-spread tuberculosis epidemics, whereas TbD1-intact “ancestral” lineages tend to be restricted to specific geographical areas, such as South India and South East Asia (L1) or East Africa (L7). By constructing and characterizing a panel of recombinant TbD1-knock-in and knock-out strains and comparison with clinical isolates, here we show that deletion of TbD1 confers to Mtb a significant increase in resistance to oxidative stress and hypoxia, which correlates with enhanced virulence in selected cellular, guinea pig and C3HeB/FeJ mouse infection models, the latter two mirroring in part the development of hypoxic granulomas in human disease progression. Our results suggest that loss of TbD1 at the origin of the L2/L3/L4 Mtb lineages was a key driver for their global epidemic spread and outstanding evolutionary success.

Candida parapsilosis is an emerging opportunistic pathogen, second in frequency only to C. albicans and commonly associated with both mucosal and systemic infections. Adhesion to biotic surfaces is a key step for the development of mycoses. The C. parapsilosis genome encodes 5 predicted agglutinin-like sequence proteins and their precise role in the adhesion process still remains to be elucidated. In this study, we focused on the putative adhesin Cpar2_404800, in view of its high homology to the most important adhesion molecule in C. albicans. Two independent lineages of C. parapsilosis CPAR2_404800 heterozygous and null mutants were obtained by site-specific deletion. CPAR2_404800 mutants did not differ from wild-type strain in terms of in vitro growth or in their ability to undergo morphogenesis. However, when compared for adhesion to a biotic surface, CPAR2_404800 null mutants exhibited a marked reduction in their adhesion to buccal epithelial cells (>60% reduction of adhesion index). Reintroduction of one copy of CPAR2_404800 gene in the null background restored wild type phenotype. A murine model of urinary tract infection was used to elucidate the in vivo contribution of CPAR2_404800. A 0.5 and 1 log₁₀ reduction in colony forming unit numbers (per gram) was observed respectively in bladder and kidneys obtained from mice infected with null mutant compared to wild-type infected ones. Taken together, these findings provide the first evidence for a direct role of CPAR2_404800 in C. parapsilosis adhesion to host surfaces and demonstrate its contribution to the pathogenesis of murine urinary candidiasis.

Candida orthopsilosis is a human fungal pathogen responsible for a wide spectrum of symptomatic infections. Evidence suggests that C. orthopsilosis is mainly susceptible to azoles, the most extensively used antifungals for treatment of these infections. However, fluconazole-resistant clinical isolates are reported. This study evaluated the contribution of a single amino acid substitution in the azole target CoErg11 to the development of azole resistance in C. orthopsilosis. C. orthopsilosis clinical isolates (n = 40) were tested for their susceptibility to azoles and their CoERG11 genes were sequenced. We used a SAT1 flipper-driven transformation to integrate a mutated CoERG11 allele in the genetic background of a fluconazole-susceptible isolate. Susceptibility testing revealed that 16 of 40 C. orthopsilosis clinical isolates were resistant to fluconazole and to at least one other azole. We identified an A395T mutation in the CoERG11 coding sequence of azole-resistant isolates only that resulted in the non-synonymous amino acid substitution Y132F. The SAT1 flipper cassette strategy led to the creation of C. orthopsilosis mutants that carried the A395T mutation in one or both CoERG11 alleles (heterozygous or homozygous mutant, respectively) in an azole-susceptible genetic background. We tested mutant strains for azole susceptibility and for hot-spot locus heterozygosity. Both the heterozygous and the homozygous mutant strains exhibited an azole-resistant phenotype. To the best of our knowledge, these findings provide the first evidence that the CoErg11 Y132F substitution confers multi-azole resistance in C. orthopsilosis.

Agglutinin like sequence (Als) cell-wall proteins play a key role in adhesion and virulence of Candida species. Compared to the well-characterized Candida albicans ALS genes, little is known about ALS genes in the Candida parapsilosis species complex. Three incomplete ALS genes were identified in the genome sequence for Candida orthopsilosis strain 90–125 (GenBank assembly ASM31587v1): CORT0C04210 (named CoALS4210), CORT0C04220 (CoALS4220) and CORT0B00800 (CoALS800). To complete the gene sequences, new data were derived from strain 90–125 using Illumina (short-read) and Oxford Nanopore (long-read) methods. Long-read sequencing analysis confirmed the presence of 3 ALS genes in C. orthopsilosis 90–125 and resolved the gaps located in repetitive regions of CoALS800 and CoALS4220. In the new genome assembly (GenBank PQBP00000000), the CoALS4210 sequence was slightly longer than in the original assembly. C. orthopsilosis Als proteins encoded features well-known in C. albicans Als proteins such as a secretory signal peptide, N-terminal domain with a peptide-binding cavity, amyloid-forming region, repeated sequences, and a C-terminal site for glycosylphosphatidylinositol anchor addition that, in yeast, suggest localization of the proteins in the cell wall. CoAls4210 and CoAls800 lacked the classic C. albicans Als tandem repeats, instead featuring short, imperfect repeats with consensus motifs such as SSSEPP and GSGN. Quantitative RT-PCR showed differential regulation of CoALS genes by growth stage in six genetically diverse C. orthopsilosis clinical isolates, which also exhibited length variation in the ALS alleles, and strain-specific gene expression patterns. Overall, long-read DNA sequencing methodology was instrumental in generating an accurate assembly of CoALS genes, thus revealing their unconventional features and first insights into their allelic variability within C. orthopsilosis clinical isolates.

Matrix assisted laser desorption ionization time of flight (MALDI-TOF) is a powerful analytical tool that has revolutionized microbial identification. Routinely used for bacterial identification, MALDI-TOF has recently been applied to both yeast and filamentous fungi, confirming its pivotal role in the rapid and reliable diagnosis of infections. Subspecies-level identification holds an important role in epidemiological investigations aimed at tracing virulent or drug resistant clones. This review focuses on present and future applications of this versatile tool in the clinical mycology laboratory.