RESUMO
The ability to sense and respond to host defenses is essential for pathogen survival. Some mechanisms involve two-component systems (TCS) that respond to host molecules, such as antimicrobial peptides (AMPs) and activate specific gene regulatory pathways to aid in survival. Alongside TCSs, bacteria coordinate cell division proteins, chaperones, cell wall sortases and secretory translocons at discrete locations within the cytoplasmic membrane, referred to as functional membrane microdomains (FMMs). In Group A Streptococcus (GAS), the FMM or "ExPortal" coordinates protein secretion, cell wall synthesis and sensing of AMP-mediated cell envelope stress via the LiaFSR three-component system. Previously we showed GAS exposure to a subset of AMPs (α-defensins) activates the LiaFSR system by disrupting LiaF and LiaS co-localization in the ExPortal, leading to increased LiaR phosphorylation, expression of the transcriptional regulator SpxA2, and altered GAS virulence gene expression. The mechanisms by which LiaFSR integrates cell envelope stress with responses to AMP activity and virulence are not fully elucidated. Here, we show the LiaFSR regulon is comprised of genes encoding SpxA2 and three membrane-associated proteins: a PspC domain-containing protein (PCP), the lipoteichoic acid-modifying protein LafB and the membrane protein insertase YidC2. Our data show phosphorylated LiaR induces transcription of these genes via a conserved operator, whose disruption attenuates GAS virulence and increases susceptibility to AMPs in a manner primarily dependent on differential expression of SpxA2. Our work expands understanding of the LiaFSR regulatory network in GAS and identifies targets for further investigation of mechanisms of cell envelope stress tolerance contributing to GAS pathogenesis.
RESUMO
Fungal infections are being caused by a broadening spectrum of fungi, yet in many cases, identification to the species level is required for proper antifungal selection. We investigated the fungal intergenic spacer (IGS) sequence in combination with nanopore sequencing for fungal identification. We sequenced isolates from two Cryptococcus species complexes, C. gattii and C. neoformans, which are the main pathogenic members of this genus, using the Oxford Nanopore Technologies MinION device and Sanger sequencing. There is enough variation within the two complexes to argue for further resolution into separate species, which we wanted to see if nanopore sequencing could detect. Using the R9.4.1 flow cell, IGS sequence identities averaged 99.57% compared to Sanger sequences of the same region. When the newer R10.3 flow cell was used, accuracy increased to 99.83% identity compared to the same Sanger sequences. Nanopore sequencing errors were predominantly in regions of homopolymers, with G homopolymers displaying the largest number of errors and C homopolymers displaying the least. Phylogenetic analysis of the nanopore- and Sanger-derived sequences resulted in indistinguishable trees. Comparison of average percent identities between the C. gattii and C. neoformans species complexes resulted in only a 74 to 77% identity between the two complexes. Sequencing using the nanopore platform could be completed in less than an hour, and samples could be multiplexed in groups as large as 24 sequences in a single run. These results suggest that sequencing the IGS region using nanopore sequencing could be a potential new molecular diagnostic strategy.
