c High-throughput RNA sequencing technology has found the 5= untranslated region of sarA to contain two putative small RNAs (sRNAs), designated teg49 and teg48. Northern blot analysis disclosed that teg49 and teg48 were detectable within the P3-P1 and P1 sarA promoter regions, respectively. Focusing on teg49, we found that this sRNA, consisting of 196 nucleotides, is transcribed in the same direction as the sarA P3 transcript. The expression of both P3 and teg49 transcripts is dependent on sigB and cshA, which encodes a DEAD box RNA helicase. Within the sRNA teg49, there are two putative hairpin-loop structures, HP1 and HP2. Transversion mutation of the HP1 loop produced a smaller amount of sarA P3 and P2 transcripts and SarA protein than the corresponding HP1 stem and the HP2 stem and loop mutations, leading to lower RNAII transcription and derepression of aur transcription. The HP1 loop mutant also exhibited less biofilm formation than the parental and complemented strains. Complementation with shuttle plasmid pEPSA5 carrying teg49 was able to reestablish sarA P3 and P2 transcription and augment RNAII expression in the HP1 loop mutant. We thus conclude that teg49, embedded within the extended promoter regions of sarA, is modulated by sigB and cshA and plays an important trans-acting role in modulating the transcription and ensuing expression of sarA.

To elucidate the molecular mechanisms controlling the expression of the hypha-specific adhesin gene HWP1 of Candida albicans, its promoter was dissected and analyzed using a green fluorescent protein reporter gene. A 368-bp region, the HWP1 control region (HCR), was critical for activation under hypha-inducing conditions and conferred developmental regulation to a heterologous ENO1 promoter. A more distal region of the promoter served to amplify the level of promoter activation. Using gel mobility shift assays, a 249-bp subregion of HCR, HCRa, was found to bind at least four proteins from crude extracts of yeasts and hyphae with differing binding patterns dependent on cell morphology. Four proteins with DNA binding activities were identified by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis after separation by anion-exchange and heparin-Sepharose chromatography. One protein with high similarity to Nhp6, an HMG1 family member in Saccharomyces cerevisiae, and another with weak similarity to an HMG-like condensation factor from Physarum polycephalum implicated changes in chromatin structure as a critical process in hypha-specific gene regulation. Proteins with strong homology to histones were also found. These studies are the first to identify proteins that bind to a DNA segment that confers developmental gene regulation in C. albicans and suggest a new model for hypha-specific gene regulation.Candida albicans is an opportunistic fungal pathogen that rapidly transitions between yeast, true hyphal, and pseudohyphal growth forms in response to a number of environmental stimuli, including temperature, pH, and medium composition (15,19,21). Changes in the external environment of C. albicans are known to be accompanied by changes in the genetic expression patterns of morphology-specific transcripts, leading in turn to the activation and deactivation of genetic pathways that are believed to underlie the multiple interactions of C. albicans with its mammalian hosts, including the colonization and invasion of tissue and resistance to host defenses (16,28).The true hyphal morphology of C. albicans (43) is notable for its enhanced adherence in comparison to yeast forms (40), and this is reflected in the presence of hypha-specific proteins that promote the invasion of and adherence to host surfaces. One such protein, Hwp1 (hyphal wall protein 1), is a glycosylphosphatidylinositol-linked cell wall protein that is abundantly expressed when C. albicans is in hyphal growth mode and is absent or barely detectable in the yeast morphology (41). Hwp1 is incorporated into the cell wall at the tips of apically growing hyphae, limiting its presence to hyphal surfaces (39).Hwp1 has been shown to play a key role in the attachment of C. albicans to human buccal epithelial cells by forming covalent bonds through its function as a substrate for epithelial transglutaminases (40). HWP1 gene expression is regulated at the level of mRNA. Northern blot analysis reveals abundant steady-state levels of HWP1 mRNA during hyphal growt...

Expression of virulence factors in is regulated by a wide range of transcriptional regulators, including proteins and small RNAs (sRNAs), at the level of transcription and/or translation. The locus consists of three overlapping transcripts generated from three distinct promoters, all containing the open reading frame (ORF). The 5' untranslated regions (UTRs) of these transcripts contain three separate regions ∼711, 409, and 146 nucleotides (nt) upstream of the translation start, the functions of which remain unknown. Recent transcriptome-sequencing (RNA-Seq) analysis and subsequent characterization indicated that two sRNAs, teg49 and teg48, are processed and likely produced from the P3 and P1 transcripts of the locus, respectively. In this report, we utilized a variety of promoter mutants and and mutants to ascertain the contributions of these factors to the generation of teg49. We also defined the transcriptional regulon of teg49, including virulence genes not regulated by SarA. Phenotypically, teg49 did not impact biofilm formation or affect overall SarA expression significantly. Comparative analyses of RNA-Seq data between the wild-type, teg49 mutant, and mutant strains indicated that ∼133 genes are significantly upregulated while 97 are downregulated in a teg49 deletion mutant in a-independent manner. An abscess model of skin infection indicated that the teg49 mutant exhibited a reduced bacterial load compared to the wild-type Overall, these results suggest that teg49 sRNA has a regulatory role in target gene regulation independent of SarA. The exact mechanism of this regulation is yet to be dissected.

bThe toxin MazF sa in Staphylococcus aureus is a sequence-specific endoribonuclease that cleaves the majority of the mRNAs in vivo but spares many essential mRNAs (e.g., secY mRNA) and, surprisingly, an mRNA encoding a regulatory protein (i.e., sarA mRNA). We hypothesize that some mRNAs may be protected by RNA-binding protein(s) from degradation by MazF sa . Using heparin-Sepharose-enriched fractions that hybridized to sarA mRNA on Northwestern blots, we identified among multiple proteins the DEAD box RNA helicase CshA (NWMN_1985 or SA1885) by mass spectroscopy. Purified CshA exhibits typical RNA helicase activities, as exemplified by RNA-dependent ATPase activity and unwinding of the DNA-RNA duplex. A severe growth defect was observed in the cshA mutant compared with the parent when grown at 25°C but not at 37°C. Activation of MazF sa in the cshA mutant resulted in lower CFU per milliliter accompanied by a precipitous drop in viability (ϳ40%) compared to those of the parent and complemented strains. NanoString analysis reveals diminished expression of a small number of mRNAs and 22 small RNAs (sRNAs) in the cshA mutant versus the parent upon MazF sa induction, thus implying protection of these RNAs by CshA. In the case of the sRNA teg049 within the sarA locus, we showed that the protective effect was likely due to transcript stability as revealed by reduced half-life in the cshA mutant versus the parent. Accordingly, CshA likely stabilizes selective mRNAs and sRNAs in vivo and as a result enhances S. aureus survival upon MazF sa induction during stress. D iscovered first as "addiction modules" in plasmids (1, 2), toxin-antitoxin (TA) systems have subsequently been found in the chromosomes of many pathogenic and nonpathogenic bacteria and Archaea (3-8). There are three types of TA systems, including RNA-RNA and protein-RNA systems (types I and III, respectively) and protein-protein systems (type II) (9). Chromosome-borne type II TA modules (9) are ubiquitous, with the small labile antitoxin binding the stable toxin to form an inert complex (5, 8). Upon stress (e.g., antibiotic, oxidation, or thymidine, or amino acid starvation) (10-15), the labile antitoxin will be degraded by ClpP in complex with specific adaptors (16,17), thus unleashing the toxin to act on its target, which can be mRNA or other targets in the transcription/translation machinery (5). Additionally, there is accumulating evidence that TA modules may have an important role in stress physiology and quality control of gene expression by reducing production of proteins not essential to bacterial survival (5).Salmonella enterica serovar Typhimurium (18) and Mycobacterium tuberculosis (19) have at least 11 and 88 type II TA modules, respectively, some of which are conserved in other pathogenic bacteria but absent from nonpathogenic species, suggesting that TA modules are critical to the virulence of these strains. In fact, the toxin-antitoxin genes sehAB in S. Typhimurium play a critical role in survival inside host cells (18).In Escherichia coli, the...

The mechanisms that fungi use to co-regulate subsets of genes specifically associated with morphogenic states represent a basic unsolved problem in fungal biology. Candida albicans is an important model of fungal differentiation both for rapid interconversion between yeast and hyphal growth forms and for white/opaque switching mechanisms. The Sundstrom lab is interested in mechanisms regulating hypha-specific expression of adhesin genes that are critical for C. albicans hyphal growth phenotypes and pathogenicity. Early studies on hypha-specific genes such as HWP1 and ALS3 reported 5’ intergenic regions that are larger than those typically found in an average promoter and are associated with hypha-specific expression. In the case of HWP1, activation and repression involves a 368 bp region, denoted the HWP1 control region (HCR), located 1410 bp upstream of its transcription start site. In previous work we showed that HCR confers developmental regulation to a heterologous ENO1 promoter, indicating that HCR by itself contains sufficient information to couple gene expression to morphology. Here we show that the activation and repression mediated by HCR are localized to distinct HCR regions that are targeted by the transcription factors Nrg1p and Efg1p. The finding that Efg1p mediates both repression via HCR under yeast morphological conditions and activation conditions positions Efg1p as playing a central role in coupling HWP1 expression to morphogenesis through the HCR region. These localization studies revealed that the 120 terminal base pairs of HCR confer Efg1p-dependent repressive activity in addition to the Nrg1p repressive activity mediated by DNA upstream of this subregion. The 120 terminal base pair subregion of HCR also contained an initiation site for an HWP1 transcript that is specific to yeast growth conditions (HCR-Y) and may function in the repression of downstream DNA. The detection of an HWP1 mRNA isoform specific to hyphal growth conditions (HWP1-H) showed that morphology-specific mRNA isoforms occur under both yeast and hyphal growth conditions. Similar results were found at the ALS3 locus. Taken together, these results, suggest that the long 5’ intergenic regions upstream of hypha-specific genes function in generating mRNA isoforms that are important for morphology-specific gene expression. Additional complexity in the HWP1 promoter involving HCR-independent activation was discovered by creating a strain lacking HCR that exhibited variable HWP1 expression during hyphal growth conditions. These results show that while HCR is important for ensuring uniform HWP1 expression in cell populations, HCR independent expression also exists. Overall, these results elucidate HCR-dependent mechanisms for coupling HWP1-dependent gene expression to morphology uniformly in cell populations and prompt the hypothesis that mRNA isoforms may play a role in coupling gene expression to morphology in C. albicans.