Salmonella enterica serovar Typhimurium (S. Tm) is a major cause of foodborne diarrhea. However, in healthy individuals, the microbiota typically restricts the growth of incoming pathogens, a protective mechanism termed colonization resistance (CR). To circumvent CR, Salmonella strains can utilize private nutrients that remain untapped by the resident microbiota. However, the metabolic pathways and environmental niches promoting pathogen growth are still not completely understood. Here, we investigate the significance of the gfr operon in gut colonization of S. Tm, which is essential for the utilization of fructoselysine (FL) and glucoselysine (GL). These Amadori compounds are present in heated foods with high protein and carbohydrate contents. We detected FL in both mouse chow and the intestinal tract of mice and showed that gfr mutants are attenuated during the initial phase of colonization in the murine model. Experiments in gnotobiotic mice and competition experiments with Escherichia coli suggest that gfr-dependent fitness advantage is context-dependent. We conclude that dietary Amadori products like FL can support S. Tm gut colonization, depending on the metabolic capacities of the microbiota.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health burden, especially with the increasing prevalence of drug-resistant strains. There is an urgent need for new therapeutics that act via alternative mechanisms. Autophagy, a vital cell-autonomous defense process, allows macrophages to degrade intracellular pathogens such as Mtb and has gained attention as a potential target for host-directed therapy. In this study, we conducted a high-content imaging screen of herb-derived compounds to identify autophagy inducers in RAW264.7 macrophages. Panduratin A (NPA), a natural compound from Boesenbergia rotunda, was found to potently induce autophagy. NPA promoted autophagic vacuole formation in a dose-dependent fashion at low micromolar levels. Its autophagy-inducing effect was validated using RFP-GFP-LC3 dual fluorescence assays and immunoblotting in the presence of bafilomycin A1. Further mechanistic analysis revealed that NPA activates autophagy through AMPK activation, independent of mTOR inhibition. Importantly, NPA significantly promoted intracellular Mtb clearance and increased colocalization of Mtb with autophagosomes and lysosomes, in a manner dependent on Beclin-1. These findings highlight NPA as a potent enhancer of macrophage antimicrobial responses via autophagy, supporting its potential as a candidate for host-directed adjunctive therapy against TB.

Sneathia vaginalis is a common component of the vaginal microbiome and is emerging as a marker for preterm birth. It produces the cytopathogenic toxin A (CptA), which is capable of lysing human red blood cells and permeabilizing epithelial cells. However, the role of CptA and other potential virulence factors in pathogenesis has been difficult to characterize due to the lack of genetic tools for targeted deletion in S. vaginalis. The objective of this study was to create the first isogenic gene deletion mutant in S. vaginalis. We chose the cptA gene as a target for deletion because of its role in virulence. We characterized the restriction-modification profile in S. vaginalis to increase the chances that exogenous DNA would resist restriction digestion, and we identified an antibiotic resistance cassette that is functional in this species. We identified a genetic locus encoding a Dam methylase and a restriction endonuclease with DpnII-like activity in S. vaginalis strain SN35. By convention, this newly described restriction endonuclease would be named SvaSI for S. vaginalis SN35. Using plasmid DNA purified from a Dam+ E. coli strain to evade SvaSI restriction, we successfully replaced cptA with an erythromycin resistance cassette encoding the ermF and ermAM genes, creating the first genetically engineered deletion mutation in this species. Results revealed that CptA is necessary for the hemolytic and cytopathogenic activities of S. vaginalis. This work is a resource that lays the foundation for the development of additional genetic tools for S. vaginalis and facilitates the characterization of additional genes in this emerging pathogen.

By regulating the assortment and abundance of its virulence factors at different anatomic sites, the group A Streptococcus (GAS) can cause a range of human diseases. The Fas regulatory system is encoded by a four-gene locus, fasBCAX, with fasX encoding the FasX small regulatory RNA effector molecule. FasX post-transcriptionally regulates target mRNAs through well-characterized mechanisms. Less characterized are the layers of regulation that occur upstream of FasX activity, such as how the products of the fasBCA genes enhance FasX abundance 100-fold. Here, we present data consistent with FasBCA forming a three-component regulatory system, with FasBC being sensor kinase-like proteins that, upon recognizing one or more signals, heterodimerize and phosphorylate FasA, with phosphorylated FasA binding to the fasX promoter and inducing transcription. We identified key amino acids involved in phosphate flow, including H246 of FasC and D60 of FasA, and demonstrated that certain domains (e.g., the kinase domain of FasC) are dispensable for activity. Additionally, we show that a proteinaceous factor within human plasma activates the Fas system. This work represents the first molecular analysis of the Fas proteins which, by modulating FasX levels, play a critical role in the ability of GAS to coordinately regulate virulence factor production.

Most bacterial populations exhibit phenotypic heterogeneity to increase fitness in rapidly changing environmental conditions. Myxococcus xanthus is an environmental bacterium that displays pronounced phenotypic heterogeneity in its complex lifecycle. Under nutrient limitation, M. xanthus produces a specialized biofilm in which cells segregate into two spatially distinct fates: fruiting bodies filled with spores and a persister-like peripheral rod population. Little is known about the regulatory mechanisms controlling peripheral rods. To begin to investigate this cell fate segregation mechanism, we focused on the EspAC signaling system, which controls the accumulation of MrpC, a central transcription factor necessary to induce fruiting body formation. Single-cell reporters and in situ confocal microscopy demonstrated that expression of the esp genes is enriched in the peripheral rods. We identified three transcription factors necessary for espAC transcriptional control: MrpC, FruA, a transcription factor that coordinates sporulation within fruiting bodies, and the xenobiotic response element, Xre0228. We demonstrate that MrpC directly activates espA and espC; FruA represses espC but not espA; and Xre0228 activates espA but represses espC. These genetic interactions fit common network motifs that promote or stabilize phenotypic heterogeneity. We propose a model by which cell fate segregation is directed, stabilized, and tuned to environmental conditions.

Bacteria have evolved complex conditional pathways that respond to environmental stresses and signals. We use conjugation in Mycobacterium smegmatis to identify contact-recognition and response pathways that mediate interactions between donor and recipient cells. Contact with a compatible donor cell initiates a response in the recipient that requires the ESX-1 secretion system and subsequently activates the dormant ESX-4 secretion system. The links of this signal transduction pathway, the mechanism of coordination and dependency between ESX-1 and ESX-4 secretion systems, are unknown. Previous studies identified SigM as a cell-contact responsive sigma factor dedicated to activating ESX-4. WhiB proteins are iron-sulfur-binding stress-response transcription factors exclusively found in Actinobacteria. WhiB6 has been shown to regulate ESX-1 associated gene expression in other mycobacteria. Here, we show that WhiB6 is required both for conjugation and for transducing cell-contact dependent signaling in the recipient cell. Our RNA-seq, ChIP-seq, and proteomic profiling data define a WhiB6 regulon that supports conjugative cell-cell interaction. The WhiB6 regulon includes genes encoding ESX-1, ESX-4, SigM, as well as dispersed operons that likely support ESX secretion. Our data demonstrate that WhiB6 is epistatic to SigM and ESX-4 in this signal transduction pathway. This work shows that WhiB6 functions as a signal transduction node in recipient cells: it coordinates the expression of two ESX systems and it also induces uncharacterized proteins that collectively constitute a complete secretion response to recipient contact with a donor cell.

The lipid phosphatase Fig4 is conserved in all eukaryotes and is associated with human neurological diseases for which there are currently no specific therapies. Fig4 functions in both the production and turnover of its lipid substrate, PI3,5P2, through participation in the Fab1-Vac14-Fig4 complex with its opposing kinase Fab1. The molecular mechanisms through which Fig4 influences PI3,5P2 production are not fully understood but are believed to require Fig4 binding to the scaffold protein Vac14. We unexpectedly found that multiple Fig4 disease-related mutants that are impaired in binding to the Fab1-Vac14-Fig4 complex dominantly confer tolerance to rapamycin, an inhibitor of the Target of Rapamycin Complex 1 (TORC1), when expressed in Saccharomyces cerevisiae. Fig4-dependent rapamycin tolerance is conferred under moderate heat stress, independent of Vac14 and Fig4 catalytic activity. Conversely, expression of catalytically dead Fig4 that binds stably to the Fab1-Vac14-Fig4 complex enhances rapamycin sensitivity. We propose that Fig4 disease-related mutants alter TORC1 signaling through gain of function under these conditions through an abnormal or sustained interaction with an unknown factor, perhaps by altering PI3,5P2 production. Investigation of the mechanisms whereby Fig4 mutants alter rapamycin tolerance may provide new insights into Fig4 molecular functions with potential relevance for Fig4-related diseases.

Phospholipid synthesis in Firmicute bacteria differs markedly from that of the paradigm Escherichia coli pathway in that acyl phosphates are a key intermediate. Acyl phosphates are required for the first acylation step of the phospholipid synthesis pathway catalyzed by the PlsY acyltransferase and are synthesized by two different pathways. In the absence of exogenous fatty acids, de novo synthesized acyl-acyl carrier protein (ACP) species are converted to acyl phosphates by the PlsX acyl-ACP: phosphate acyltransferase, which transfers the acyl chain from ACP to inorganic phosphate. When exogenous fatty acids are present, these acids are converted to acyl phosphates by the FakAB fatty acid kinase and can be converted to acyl-ACPs via PlsX. The active kinase is composed of the ATP-requiring FakA subunit and a FakB fatty acid binding protein, which acts to present the fatty acid carboxyl group to the FakA kinase active site. In all Firmicutes examined to date, multiple FakB species are present. Staphylococcus aureus has two, whereas Streptococcus pneumoniae has three, whereas Enterococcus faecalis encodes four FakB proteins. We report the fatty acid preferences of these proteins obtained by use of mutant strains lacking each FakB or all possible combinations of three FakB deletions, plus a strain lacking all four FakB proteins. We also report the phenotype of a ∆fakA strain and of a ∆fakA bypass suppressor mutant, plus the first indication of a role of the FakAB pathway in recycling of acyl chains.

The insertion sequence IS200 is widely distributed in Eubacteria. Despite its prevalence, IS200 does not appear to be mobile and as such is considered an ancestral component of bacterial genomes. Previous work in Salmonella enterica revealed that the IS200 tnpA transcript is processed to form a small, highly structured RNA (5'tnpA) that participates in the posttranscriptional control of invF expression, encoding a key transcription factor in this enteropathogen's invasion regulon. To further examine the scope of 5'tnpA transcript integration into Salmonella gene expression networks, we performed comparative RNA-seq, revealing the differential expression of over 200 genes in a Salmonella SL1344 5'tnpA disruption strain. This includes the genes for the master regulators of both invasion and flagellar regulons (HilD and FlhDC, respectively), plus genes involved in cysteine biosynthesis and an operon (phsABC) encoding a thiosulfate reductase complex. These expression changes were accompanied by an 80-fold increase in Salmonella invasion of HeLa cells. Follow-up experimentation suggested an additional direct target of 5'tnpA to be the small RNA PinT, which has previously been shown to be a negative regulator of invasion genes through its inhibitory action on key transcription factors governing the Salmonella pathogenicity island 1 regulon. This study provides a powerful new example of bacterial transposon domestication that is based not on the production/use of a regulatory protein or regulatory DNA sequences, but on the function of a transposon-derived small RNA.

The mechanisms by which cells respond to growth inhibitory redox-cycling agents is only partially understood. In Escherichia coli K12, the IscR regulon, which includes the ISC and SUF Fe-S cluster biogenesis machineries, is differentially expressed in response to these agents. Here, we report how one redox-cycling agent, phenazine methosulfate (PMS), regulates IscR activity via its [2Fe-2S] cluster cofactor. A direct role for IscR in mediating the response to PMS was inferred from the PMS-dependent weakening of [2Fe-2S]-IscR binding to an isc operon type 1 DNA site in vitro. This decrease in DNA binding was attributed to the accompanying oxidation of its [2Fe-2S] cluster. Exposure of anaerobic cultures to PMS leads to increased isc expression, as expected from IscR cluster oxidation and impaired binding to type 1 sites in the isc promoter. However, this same anaerobic PMS treatment did not change expression of type 2 site promoters, such as suf, which require IscR that lacks an Fe-S cluster (apo-IscR) for effective transcriptional regulation. In contrast, PMS exposure under aerobic conditions significantly increased both isc and suf expression, indicating the formation of both [2Fe-2S]-IscR and apo-IscR. This effect was partially attributed to superoxide generation by PMS under aerobic conditions, as evidenced by a superoxide dismutase-deficient mutant showing a modest impact on isc and suf expression. Together, these findings provide new insights into redox-cycling dependent regulation of IscR activity and highlight the distinct activities of apo-IscR, [2Fe-2S]-IscR and [2Fe-2S]-IscR in controlling the IscR regulon.

Streptococcus agalactiae (group B Streptococcus; GBS) is a leading cause of neonatal sepsis and meningitis. Despite advances in molecular microbiology, GBS genome engineering remains laborious due to inefficient mutagenesis protocols. Here, we report a versatile and rapid Cas12a-based toolkit for GBS genetic manipulation. We developed two shuttle plasmids-pGBSedit for genome editing and pGBScrispri for inducible CRISPR interference-derived from an Enterococcus faecium system and optimized for GBS. Using these tools, we achieved targeted gene insertions, markerless deletions, and efficient, template-free mutagenesis via alternative end-joining repair. Furthermore, a catalytically inactive dCas12a variant enabled inducible gene silencing, with strand-specific targeting effects. The system demonstrated broad applicability across multiple GBS strains and minimal off-target activity, as confirmed by whole-genome sequencing. In benchmarking, template-less Cas12a mutagenesis yielded sequence-confirmed mutants in ~7 days and homology-directed edits in ~7-14 days; aTC-resistant colonies arose at ~10 of uninduced CFU, and 27%-65% of resistant clones carried the intended homology-directed edit depending on locus and homology arm length (e.g., ~27% markerless deletion; ~35% insertion; 65% with 1 kb arms). These workflows provide a rapid alternative to temperature-sensitive plasmid mutagenesis protocols that typically require ≥ 4 weeks. This Cas12a-based platform offers an efficient, flexible, and scalable approach to genetic studies in GBS, facilitating functional genomics and accelerating pathogenesis research.

Clostridium perfringens is a gram-positive, anaerobic, spore-forming bacterial pathogen of humans and animals. C. perfringens also produces type IV pili (T4P) and has two complete sets of T4P-associated genes, one of which has been shown to produce surface pili needed for cell adherence. One hypothesis about the second set of T4P genes is that they comprise a type II secretion system (TTSS) like those found in gram-negative bacteria, but for gram-positive bacteria, the TTSS would aid transit across the thick peptidoglycan (PG) layer. The secretome of mutants lacking type IV pilins was examined, and a single protein, BsaC (CPE0517), was identified as being dependent on pilin PilA3 for secretion. The bsaC gene is in an operon with genes encoding a SipW signal peptidase and two putative biofilm matrix proteins, BsaA and BsaB, both of which have remote homology to Bacillus subtilis biofilm protein TasA. Since BsaA forms long oligomers that are secreted, we analyzed BsaA monomer interactions with de novo modeling. These models projected that the monomers formed isopeptide bonds as part of a donor strand exchange process. Mutations in residues predicted to form the isopeptide bonds led to the loss of oligomerization, supporting an exchange and lock mechanism, and isopeptide bonds were detected by mass spectrometry methods. Phylogenetic analysis showed the BsaA family of proteins is widespread among bacteria and archaea, but only a subset is predicted to form isopeptide bonds.

BosR, the sole member of the ferric uptake regulator (FUR) family in Borrelia burgdorferi, is essential for the spirochete's transcriptional adaptation to the mammalian host environment. Although best known for activating rpoS and establishing the mammalian-phase RpoS regulon, BosR originally was linked to regulation of genes involved in B. burgdorferi's oxidative stress response. Here, we show that BosR governs gene expression through both RpoS-dependent and RpoS-independent mechanisms under in vitro and mammalian host-adapted conditions. Using RNA-seq and a DNA-binding-defective BosR-R39A mutant, we demonstrate that DNA binding is essential for BosR's global regulatory functions. BosR activates rpoS, promotes RpoS-dependent gene regulation, and independently modulates a distinct set of genes involved in a variety of cellular functions, including genome maintenance, chemotaxis, and virulence. Notably, canonical oxidative stress response genes previously attributed to BosR were not differentially expressed in ΔbosR strains in vitro or in mammals. Despite its broad regulatory scope, BosR does not recognize a single, conserved DNA-binding motif, suggesting that DNA occupancy is influenced by local sequence context or DNA topology. Our findings support a bifunctional model in which BosR collaborates with RNA polymerase (RNAP)-RpoS holoenzyme to activate and repress RpoS-regulated genes, while functioning in a FUR-like manner to control RpoD-dependent genes independently of RNAP interaction.

Flavin mononucleotide (FMN) riboswitches are RNA-based regulatory elements found in many bacteria. FMN riboswitches control genes responsible for the biosynthesis and transport of riboflavin (rib genes). Riboflavin (vitamin B) is the precursor of the flavoenzyme cofactors FMN and flavin adenine dinucleotide (FAD), and it is FMN (not riboflavin or FAD) that is perceived by FMN riboswitches as a signal with regard to flavin homeostasis. When FMN levels are adequate, expression of rib genes is shut down by FMN riboswitches. The bifunctional protein RibR from the Gram-positive bacterium Bacillus subtilis contains an enzymatic and a regulatory part and is induced when cells encounter specific sulfur sources. Under these conditions, B. subtilis RibR binds to FMN riboswitches, overrides their genetic decisions, and stimulates rib gene expression. In B. subtilis, the objective of this RibR-mediated superordinate control is to link sulfur metabolism to riboflavin metabolism. B. subtilis RibR was previously the only known riboswitch-modulating protein. We now report on a similar but monofunctional protein from Bacillus amyloliquefaciens. RibR from B. amyloliquefaciens contains a regulatory/RNA-binding part only, and ribR expression is not stimulated by sulfur sources but by the disulfide-generating and oxidative stress-inducing compound diamide. RibR-like regulator proteins may be more widespread than anticipated and apparently have evolved to connect riboswitch-controlled pathways to other pathways.

Candida albicans is a commensal microbe and opportunistic human pathogen. Candida yeast are recognized and taken up by macrophages via phagocytosis. Macrophage surface receptors bind to specific components of the Candida cell wall. Following phagocytosis, Candida can respond to the host's intracellular environment by switching from a yeast to a hyphal morphology facilitating escape from macrophages and allowing subsequent invasion of host tissues. Various disruptions of Candida's ability to form hyphae have been shown to reduce virulence and fitness in the host. Our previous work concluded that Candida albicans cells lacking AP-2 (apm4Δ/Δ), an endocytic adaptor complex, have increased cell wall chitin and morphologically defective hyphae in vitro. Increased chitin has been correlated with decreased recognition by macrophages, possibly due to masking of cell wall β-glucan, the target for the Dectin-1 immune receptor. Here we test the virulence profiles of apm4Δ/Δ mutant, demonstrating a surprising increase in macrophage phagocytosis that does not occur due to the elevated exposure of β-glucan, highlighting the importance of cell wall components beyond chitin and glucan for macrophage engagement and uptake. Furthermore, the apm4 mutant exhibited parasitism of macrophages, surviving and proliferating within the phagosome, a phenotype that was then replicated with a well-characterized yeast locked mutant, demonstrating the further complexity of C. albicans' ability to evade macrophage responses. Finally, the combined phenotype of reduced hyphal formation but continued proliferation resulted in reduced virulence despite an equivalent burden of infection with wild-type Candida infection, as determined using a zebrafish larval model of candidiasis.

Molecular chaperones play a critical role in proteostasis by aiding the folding of newly synthesized proteins and the refolding of misfolded proteins. Cells must match the protein synthesis rate to the protein folding capacity to avoid the accumulation of unfolded proteins that can form toxic aggregates. The Hsp70 chaperone DnaK binds to ribosomes and decreases protein synthesis in the bacterial pathogen Salmonella enterica serovar Typhimurium when facing cytoplasmic Mg starvation, an infection-relevant stress that disrupts proteostasis. DnaK decreases protein synthesis independently of J-domain cochaperones and nucleotide exchange factor GrpE even though J-domain cochaperones and GrpE are required for DnaK's canonical role in protein folding and refolding. DnaK's activity contrasts with that exhibited by the bacteria-specific chaperone trigger factor, which associates with ribosomes and carries out cotranslational protein folding in Mg-abundant conditions. Under infection-relevant conditions, the master regulator of S. typhimurium virulence and Mg homeostasis PhoP promotes the expression of DnaK, but not of J-domain cochaperones, GrpE, or trigger factor, suggesting that the differential expression of chaperones and cochaperones furthers S. typhimurium pathogenesis. Hsp70 chaperones also associate with ribosomes in eukaryotic cells but instead promote protein synthesis, the opposite effect that DnaK binding to ribosomes has in bacteria. Thus, Hsp70 chaperone activity differs across growth conditions and among organisms.

The endoplasmic reticulum-Golgi intermediate compartment (ERGIC) plays a crucial role in the secretory pathway; however, its existence and function in lower eukaryotes remain largely unexamined. In this study, we identified Emp43 (SPBC4F6.05c) of Schizosaccharomyces pombe, an orthologue of human (Homo sapiens) ERGIC-53, and demonstrated its localization to an ERGIC-like compartment. The localization of Emp43 depended on its C-terminal KYL motif and oligomerization through the CC1 domain. Deletion of S. pombe emp43 resulted in significant sensitivity to MgCl and FK506, along with defects in septum integrity, indicating a role in cell wall maintenance. Further analysis identified Ssp120 of S. pombe, an orthologue of human MCFD2, as a functional partner of Emp43. Yeast two-hybrid assays confirmed a strong interaction between Emp43 and Ssp120, and both proteins co-localized within an ERGIC-like compartment. Additionally, we identified Meu17 of S. pombe, a glucan-α-1,4-glucosidase homolog, as a potential ligand for Emp43. Overexpression of Meu17 rescued MgCl sensitivity in both emp43Δ and ssp120Δ strains, while mutations in its N-linked glycosylation sites (N383, N409) or its predicted active site (D203) disrupted its septum localization and functional rescue capability. Our findings indicate that Emp43 forms a complex with Ssp120 to facilitate the transport of glycosylated proteins, such as Meu17, within an ERGIC-like compartment in fission yeast S. pombe. This study provides the first evidence of an ERGIC-like structure in S. pombe and highlights the conserved nature of ERGIC-associated mechanisms across eukaryotes.

Gα, Gβ, and Gγ-the heterotrimeric G protein subunits transmit signals from G-protein coupled receptors (GPCRs) to downstream pathways. The causative agent of Amoebiasis, Entamoeba, has been presumed to contain a GPCR signaling pathway playing a role in its encystation, phagocytosis, and motility. A Gα subunit, EhGα1, has been characterized earlier in Entamoeba histolytica , which is involved in its different pathogenic processes. Here, we have characterized its ortholog, EiGα1, in the reptilian model, Entamoeba invadens , which expresses both in trophozoites and during encystation. Silencing EiGα1 through trigger-mediated knockdown reduces efficiency and leads to improper cell aggregation and abnormal chitin wall formation during encystation. Downregulation of EiGα1 results in anomalous F-actin polymerization. EiGα1 silenced cells also exhibit loss of polarity and reduced motility. Furthermore, EiGα1 knockdown also results in decreased phagocytosis of bacteria. Our findings indicate that EiGα1 controls the expression of two vital proteins in Entamoeba-the atypical EiMAPK15 and the homeobox transcription factor EiHbox1-which modulates cyst-wall development and actin reorganization. In conclusion, our findings provide strong evidence for a GPCR signaling network in Entamoeba and highlight the essential function of the Gα subunit in stage conversion and actin cytoskeleton rearrangement.

The variant surface glycoprotein (VSG) of African trypanosomes is essential for the survival of bloodstream form parasites. These parasites undergo antigenic variation, an immune evasion strategy in which they periodically switch VSG expression from one isoform to another. The molecular processes central to the expression and regulation of the VSG are however not fully understood. In general, the regulation of gene expression in trypanosomes is largely post-transcriptional. Regulatory sequences, mostly present in the 3' UTRs, often serve as key elements in the modulation of the levels of individual mRNAs. In T. brucei VSG genes, a 16mer motif within the 3' UTR has been shown to be essential for the stability of VSG transcripts and abundant VSG expression. This motif is 100% conserved in the 3' UTRs of all transcribed and non-transcribed VSG genes. As a stability-associated sequence element, the absence of nucleotide substitutions in the 16mer is however exceptional. We therefore hypothesised that the motif is involved in other essential roles/processes besides the stability of the VSG transcripts. In this study, we demonstrate that the 100% conservation of the 16mer motif is not essential for cell viability or for the maintenance of functional VSG protein levels. We further show that the intact motif in the active VSG 3' UTR is neither required to promote VSG silencing during switching nor is it needed during differentiation from bloodstream forms to procyclic forms. Ectopic overexpression of a second VSG, however, requires the intact 16mer motif within the ectopic VSG 3' UTR to trigger silencing and exchange of the active VSG, suggesting a role for the motif in transcriptional VSG switching. The enigmatic 16mer motif therefore appears to play a dual role in transcriptional VSG switching and VSG transcript stability.

Candida dubliniensis is the most closely related species to C. albicans, one of the leading causes of fungal infections in humans. However, despite sharing many characteristics, C. dubliniensis is significantly less pathogenic. To better understand the molecular underpinnings of these dissimilarities, we focused on the regulation of filamentation, a developmental trait fundamental for host colonization. We generated a collection of 44 C. dubliniensis null mutants of transcription regulators whose orthologs in C. albicans had been previously implicated in filamentous growth. These regulators are very similar at the sequence level, but phenotypic screening identified several mutants with contrasting interspecific filamentation phenotypes beyond previously known differences. Bcr1, a well-known regulator of biofilm formation, stands out as its mutant mainly showed a filamentation defect in C. dubliniensis. Phenotypic and transcriptional characterization showed that the bcr1 defect is condition dependent and that this regulator plays a central role in the filamentation of C. dubliniensis, possibly by regulating the hyphal activator Ume6. Overall, our results suggest that several regulatory pathways are involved in the filamentation differences between C. albicans and C. dubliniensis and show that the C. dubliniensis mutant collection is a valuable resource to compare, at a molecular level, these species of medical relevance.

Heat adaptation is a multilayered universal process involving a coordinated response of general and heat-specific cellular systems and processes. Here, we demonstrate that adaptation of the plant pathogenic fungus Botrytis cinerea to mild heat stress requires both autophagy and the mitochondrial Lon1 protease. Deleting bclon1 or blocking autophagy by deleting the bcatg1 autophagy-regulating gene did not affect fungal survival at optimal temperature. Under heat stress, deletion of bclon1 induced earlier and more intense autophagy, mitochondrial malfunction, and accelerated fungal cell death. These phenomena were intensified in a bcatg1/lon1 double mutant, indicating coordinated activity of both pathways in heat adaptation. Blocking autophagy, but not bclon1, also affected mycelia growth, spore germination, as well as nuclei division and spore morphology. Our results support a cytoprotective role for autophagy downstream of mitochondria-driven death signals, possibly as a mechanism that promotes growth arrest and helps remove damaged cellular components.