A variant ECE1 allele contributes to reduced pathogenicity of Candida albicans during vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving immunopathology at the vaginal mucosa. This study aimed to determine if expression and/or processing of Ece1p differs across C. albicans isolates and whether this partly underlies differential pathogenicity observed clinically. Using a targeted sequencing approach, we determined that isolate 529L harbors a similarly expressed, yet distinct Ece1p isoform variant that encodes for a predicted functional candidalysin; this isoform was conserved amongst a collection of clinical isolates. Expression of the ECE1 open reading frame (ORF) from 529L in an SC5314-derived ece1Δ/Δ strain resulted in significantly reduced vaginopathogenicity as compared to an isogenic control expressing a wild-type (WT) ECE1 allele. However, in vitro challenge of vaginal epithelial cells with synthetic candidalysin demonstrated similar toxigenic activity amongst SC5314 and 529L isoforms. Creation of an isogenic panel of chimeric strains harboring swapped Ece1p peptides or HiBiT tags revealed reduced secretion with the ORF from 529L that was associated with reduced virulence. A genetic survey of 78 clinical isolates demonstrated a conserved pattern between Ece1p P2 and P3 sequences, suggesting that substrate specificity around Kex2p-mediated KR cleavage sites involved in protein processing may contribute to differential pathogenicity amongst clinical isolates. Therefore, we present a new mechanism for attenuation of C. albicans virulence at the ECE1 locus.

Transposase-Mediated Excision, Conjugative Transfer, and Diversity of ICE6013 Elements in Staphylococcus aureus.

ICE6013 represents one of two families of integrative conjugative elements (ICEs) identified in the pan-genome of the human and animal pathogen Staphylococcus aureus Here we investigated the excision and conjugation functions of ICE6013 and further characterized the diversity of this element. ICE6013 excision was not significantly affected by growth, temperature, pH, or UV exposure and did not depend on recA The IS30-like DDE transposase (Tpase; encoded by orf1 and orf2) of ICE6013 must be uninterrupted for excision to occur, whereas disrupting three of the other open reading frames (ORFs) on the element significantly affects the level of excision. We demonstrate that ICE6013 conjugatively transfers to different S. aureus backgrounds at frequencies approaching that of the conjugative plasmid pGO1. We found that excision is required for conjugation, that not all S. aureus backgrounds are successful recipients, and that transconjugants acquire the ability to transfer ICE6013 Sequencing of chromosomal integration sites in serially passaged transconjugants revealed a significant integration site preference for a 15-bp AT-rich palindromic consensus sequence, which surrounds the 3-bp target site that is duplicated upon integration. A sequence analysis of ICE6013 from different host strains of S. aureus and from eight other species of staphylococci identified seven divergent subfamilies of ICE6013 that include sequences previously classified as a transposon, a plasmid, and various ICEs. In summary, these results indicate that the IS30-like Tpase functions as the ICE6013 recombinase and that ICE6013 represents a diverse family of mobile genetic elements that mediate conjugation in staphylococci.IMPORTANCE Integrative conjugative elements (ICEs) encode the abilities to integrate into and excise from bacterial chromosomes and plasmids and mediate conjugation between bacteria. As agents of horizontal gene transfer, ICEs may affect bacterial evolution. ICE6013 represents one of two known families of ICEs in the pathogen Staphylococcus aureus, but its core functions of excision and conjugation are not well studied. Here, we show that ICE6013 depends on its IS30-like DDE transposase for excision, which is unique among ICEs, and we demonstrate the conjugative transfer and integration site preference of ICE6013 A sequence analysis revealed that ICE6013 has diverged into seven subfamilies that are dispersed among staphylococci.

Differential requirements of protein geranylgeranylation for the virulence of human pathogenic fungi.

Protein prenylation is a crucial post-translational modification largely mediated by two heterodimeric enzyme complexes, farnesyltransferase and geranylgeranyltransferase type-I (GGTase-I), each composed of a shared α-subunit and a unique ß-subunit. GGTase-I enzymes are validated drug targets that contribute to virulence in Cryptococcus neoformans and to the yeast-to-hyphal transition in Candida albicans. Therefore, we sought to investigate the importance of the α-subunit, RamB, and the ß-subunit, Cdc43, of the A. fumigatus GGTase-I complex to hyphal growth and virulence. Deletion of cdc43 resulted in impaired hyphal morphogenesis and thermo-sensitivity, which was exacerbated during growth in rich media. The Δcdc43 mutant also displayed hypersensitivity to cell wall stress agents and to cell wall synthesis inhibitors, suggesting alterations of cell wall biosynthesis or stress signaling. In support of this, analyses of cell wall content revealed decreased amounts of ß-glucan in the Δcdc43 strain. Despite strong in vitro phenotypes, the Δcdc43 mutant was fully virulent in two models of murine invasive aspergillosis, similar to the control strain. We further found that a strain expressing the α-subunit gene, ramB, from a tetracycline-inducible promoter was inviable under non-inducing in vitro growth conditions and was virtually avirulent in both mouse models. Lastly, virulence studies using C. albicans strains with tetracycline-repressible RAM2 or CDC43 expression revealed reduced pathogenicity associated with downregulation of either gene in a murine model of disseminated infection. Together, these findings indicate a differential requirement for protein geranylgeranylation for fungal virulence, and further inform the selection of specific prenyltransferases as promising antifungal drug targets for each pathogen.

Staphylococci on ICE: Overlooked agents of horizontal gene transfer.

Horizontal gene transfer plays a significant role in spreading antimicrobial resistance and virulence genes throughout the genus Staphylococcus, which includes species of clinical relevance to humans and animals. While phages and plasmids are the most well-studied agents of horizontal gene transfer in staphylococci, the contribution of integrative conjugative elements (ICEs) has been mostly overlooked. Experimental work demonstrating the activity of ICEs in staphylococci remained frozen for years after initial work in the 1980s that showed Tn916 was capable of transfer from Enterococcus to Staphylococcus. However, recent work has begun to thaw this field. To date, 2 families of ICEs have been identified among staphylococci - Tn916 that includes the Tn5801 subfamily, and ICE6013 that includes at least 7 subfamilies. Both Tn5801 and ICE6013 commonly occur in clinical strains of S. aureus. Tn5801 is the most studied of the Tn916 family elements in staphylococci and encodes tetracycline resistance and a protein that, when expressed in Escherichia coli, inhibits restriction barriers to incoming DNA. ICE6013 is among the shortest known ICEs, but it still includes many uncharacterized open reading frames. This element uses an IS30-like transposase as its recombinase, providing some versatility in integration sites. ICE6013 also conjugatively transfers among receptive S. aureus strains at relatively higher frequency than Tn5801. Continued study of these mobile genetic elements may reveal the full extent to which ICEs impact horizontal gene transfer and the evolution of staphylococci.