The success of Mycobacterium tuberculosis as a human pathogen is due in part to its ability to survive stress conditions, such as hypoxia or nutrient deprivation, by entering nongrowing states. In these low-metabolism states, M. tuberculosis can tolerate antibiotics and develop genetically encoded antibiotic resistance, making its metabolic adaptation to stress crucial for survival. Numerous bacteria, including M. tuberculosis, have been shown to reduce their rates of mRNA degradation under growth limitation and stress. While the existence of this response appears to be conserved across species, the underlying bacterial mRNA stabilization mechanisms remain unknown. To better understand the biology of nongrowing mycobacteria, we sought to identify the mechanistic basis of mRNA stabilization in the nonpathogenic model Mycobacterium smegmatis. We found that mRNA half-life was responsive to energy stress, with carbon starvation and hypoxia causing global mRNA stabilization. This global stabilization was rapidly reversed when hypoxia-adapted cultures were reexposed to oxygen, even in the absence of new transcription. The stringent response and RNase levels did not explain mRNA stabilization, nor did transcript abundance. This led us to hypothesize that metabolic changes during growth cessation impact the activities of degradation proteins, increasing mRNA stability. Indeed, bedaquiline and isoniazid, two drugs with opposing effects on cellular energy status, had opposite effects on mRNA half-lives in growth-arrested cells. Taken together, our results indicate that mRNA stability in mycobacteria is not directly regulated by growth status but rather is dependent on the status of energy metabolism. IMPORTANCE The logistics of tuberculosis therapy are difficult, requiring multiple drugs for many months. Mycobacterium tuberculosis survives in part by entering nongrowing states in which it is metabolically less active and thus less susceptible to antibiotics. Basic knowledge on how M. tuberculosis survives during these low-metabolism states is incomplete, and we hypothesize that optimized energy resource management is important. Here, we report that slowed mRNA turnover is a common feature of mycobacteria under energy stress but is not dependent on the mechanisms that have generally been postulated in the literature. Finally, we found that mRNA stability and growth status can be decoupled by a drug that causes growth arrest but increases metabolic activity, indicating that mRNA stability responds to metabolic status rather than to growth rate per se. Our findings suggest a need to reorient studies of global mRNA stabilization to identify novel mechanisms that are presumably responsible.

Bacteria have a remarkable ability to sense environmental changes, swiftly regulating their transcriptional and posttranscriptional machinery as a response. Under conditions that cause growth to slow or stop, bacteria typically stabilize their transcriptomes in what has been shown to be a conserved stress response. In recent years, diverse studies have elucidated many of the mechanisms underlying mRNA degradation, yet an understanding of the regulation of mRNA degradation under stress conditions remains elusive. In this review we discuss the diverse mechanisms that have been shown to affect mRNA stability in bacteria. While many of these mechanisms are transcript-specific, they provide insight into possible mechanisms of global mRNA stabilization. To that end, we have compiled information on how mRNA fate is affected by RNA secondary structures; interaction with ribosomes, RNA binding proteins, and small RNAs; RNA base modifications; the chemical nature of 5 ends; activity and concentration of RNases and other degradation proteins; mRNA and RNase localization; and the stringent response. We also provide an analysis of reported relationships between mRNA abundance and mRNA stability, and discuss the importance of stress-associated mRNA stabilization as a potential target for therapeutic development.

Regulation of gene expression is critical for Mycobacterium tuberculosis to tolerate stressors encountered during infection and for nonpathogenic mycobacteria such as Mycobacterium smegmatis to survive environmental stressors. Unlike better-studied models, mycobacteria express ∼14% of their genes as leaderless transcripts. However, the impacts of leaderless transcript structures on mRNA half-life and translation efficiency in mycobacteria have not been directly tested. For leadered transcripts, the contributions of 5′ untranslated regions (UTRs) to mRNA half-life and translation efficiency are similarly unknown. In M. tuberculosis and M. smegmatis, the essential sigma factor, SigA, is encoded by a transcript with a relatively short half-life. We hypothesized that the long 5′ UTR of sigA causes this instability. To test this, we constructed fluorescence reporters and measured protein abundance, mRNA abundance, and mRNA half-life and calculated relative transcript production rates. The sigA 5′ UTR conferred an increased transcript production rate, shorter mRNA half-life, and decreased apparent translation rate compared to a synthetic 5′ UTR commonly used in mycobacterial expression plasmids. Leaderless transcripts appeared to be translated with similar efficiency as those with the sigA 5′ UTR but had lower predicted transcript production rates. A global comparison of M. tuberculosis mRNA and protein abundances failed to reveal systematic differences in protein/mRNA ratios for leadered and leaderless transcripts, suggesting that variability in translation efficiency is largely driven by factors other than leader status. Our data are also discussed in light of an alternative model that leads to different conclusions and suggests leaderless transcripts may indeed be translated less efficiently. IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis, is a major public health problem killing 1.5 million people globally each year. During infection, M. tuberculosis must alter its gene expression patterns to adapt to the stress conditions it encounters. Understanding how M. tuberculosis regulates gene expression may provide clues for ways to interfere with the bacterium’s survival. Gene expression encompasses transcription, mRNA degradation, and translation. Here, we used Mycobacterium smegmatis as a model organism to study how 5′ untranslated regions affect these three facets of gene expression in multiple ways. We furthermore provide insight into the expression of leaderless mRNAs, which lack 5′ untranslated regions and are unusually prevalent in mycobacteria.

BackgroundHospital acquired fungal infections are defined as “never events”—medical errors that should never have happened. Systemic Candida albicans infections results in 30–50% mortality rates. Typically, adhesion to abiotic medical devices and implants initiates such infections. Efficient adhesion initiates formation of aggressive biofilms that are difficult to treat. Therefore, inhibitors of adhesion are important for drug development and likely to have a broad spectrum efficacy against many fungal pathogens. In this study we further the development of a small molecule, Filastatin, capable of preventing C. albicans adhesion. We explored the potential of Filastatin as a pre-therapeutic coating of a diverse range of biomaterials.MethodsFilastatin was applied on various biomaterials, specifically bioactive glass (cochlear implants, subcutaneous drug delivery devices and prosthetics); silicone (catheters and other implanted devices) and dental resin (dentures and dental implants). Adhesion to biomaterials was evaluated by direct visualization of wild type C. albicans or a non-adherent mutant edt1 −/− that were stained or fluorescently tagged. Strains grown overnight at 30 °C were harvested, allowed to attach to surfaces for 4 h and washed prior to visualization. The adhesion force of C. albicans cells attached to surfaces treated with Filastatin was measured using Atomic Force Microscopy. Effectiveness of Filastatin was also demonstrated under dynamic conditions using a flow cell bioreactor. The effect of Filastatin under microfluidic flow conditions was quantified using electrochemical impedance spectroscopy. Experiments were typically performed in triplicate.ResultsTreatment with Filastatin significantly inhibited the ability of C. albicans to adhere to bioactive glass (by 99.06%), silicone (by 77.27%), and dental resin (by 60.43%). Atomic force microcopy indicated that treatment with Filastatin decreased the adhesion force of C. albicans from 0.23 to 0.017 nN. Electrochemical Impedance Spectroscopy in a microfluidic device that mimic physiological flow conditions in vivo showed lower impedance for C. albicans when treated with Filastatin as compared to untreated control cells, suggesting decreased attachment. The anti-adhesive properties were maintained when Filastatin was included in the preparation of silicone materials.ConclusionWe demonstrate that Filastatin treated medical devices prevented adhesion of Candida, thereby reducing nosocomial infections.

The mechanisms and regulation of RNA degradation in mycobacteria have been subject to increased interest following the identification of interplay between RNA metabolism and drug resistance. Mycobacteria encode multiple ribonucleases that are predicted to participate in mRNA degradation and/or processing of stable RNAs. RNase E is an endoribonuclease hypothesized to play a major role in mRNA degradation due to its essentiality in mycobacteria and its role in mRNA degradation in gram-negative bacteria. Here, we defined the impact of RNase E on mRNA degradation rates transcriptome-wide in the non-pathogenic model Mycolicibacterium smegmatis. RNase E played a rate-limiting role in the degradation of at least 89% of protein-coding genes, with leadered transcripts generally being more affected by RNase E repression than leaderless transcripts. There was an apparent global slowing of transcription in response to knockdown of RNase E, suggesting that M. smegmatis regulates transcription in responses to changes in mRNA degradation. This compensation was incomplete, as the abundance of most transcripts increased upon RNase E knockdown. We assessed the sequence preferences for cleavage by RNase E transcriptome-wide in both M. smegmatis and M. tuberculosis, and found a consistent bias for cleavage in C-rich regions. Purified RNase E had a clear preference for cleavage immediately upstream of cytidines, distinct from the sequence preferences of RNase E in gram-negatives. We furthermore report a high-resolution map of mRNA cleavage sites in M. tuberculosis, which occur primarily within the RNase E-preferred sequence context, confirming RNase E as a broad contributor to M. tuberculosis transcriptome structure.

16Regulation of gene expression is critical for the pathogen Mycobacterium tuberculosis to tolerate 17 stressors encountered during infection, and for non-pathogenic mycobacteria such as 18Mycobacterium smegmatis to survive stressors encountered in the environment. Unlike better 19 studied models, mycobacteria express ~14% of their genes as leaderless transcripts. However, the 20 impacts of leaderless transcript structures on mRNA half-life and translation efficiency in 21 mycobacteria have not been directly tested. For leadered transcripts, the contributions of 5' UTRs 22 to mRNA half-life and translation efficiency are similarly unknown. In both M. tuberculosis and 23 M. smegmatis, the essential sigma factor, SigA, is encoded by an unstable transcript with a 24 relatively short half-life. We hypothesized that sigA's long 5' UTR caused this instability. To test 25 this, we constructed fluorescence reporters and then measured protein abundance, mRNA 26 abundance, and mRNA half-life. From these data we also calculated relative transcription rates. 27We found that the sigA 5' UTR confers an increased transcription rate, a shorter mRNA half-life, 28 and a decreased translation rate compared to a synthetic 5' UTR commonly used in mycobacterial 29 expression plasmids. Leaderless transcripts produced less protein compared to any of the leadered 30 transcripts. However, translation rates were similar to those of transcripts with the sigA 5' UTR, 31 and the protein levels were instead explained by lower transcript abundance. A global comparison 32 of M. tuberculosis mRNA and protein abundances failed to reveal systematic differences in 33 protein:mRNA ratios for natural leadered and leaderless transcripts, consistent with the idea that 34 variability in translation efficiency among mycobacterial genes is largely driven by factors other 35 than leader status. The variability in mRNA half-life and predicted transcription rate among our 36 constructs could not be explained by their different translation efficiencies, indicating that other 37 factors are responsible for these properties and highlighting the myriad and complex roles played 38 by 5' UTRs and other sequences downstream of transcription start sites. 39 40 41 5' UTRs can also regulate gene expression by altering mRNA turnover rates. This can be a 57 consequence of altered translation rates, as impairments to translation often lead to faster mRNA 58 decay [16][17][18][19][20][21][22]. In other cases, mRNA stability is directly affected by sRNA binding to 5' UTRs or 59 by UTR secondary structure [9,[23][24][25][26][27][28]. In E. coli, the half-life of the short-lived transcript bla can 60 be significantly increased when its native 5' UTR is replaced with the 5' UTR of ompA, a long-61 lived transcript [29][30][31]. Conversely, deletion of ompA's native 5' UTR decreased its half-life by 62 5-fold [30]. The longevity conferred by the ompA 5' UTR was attributed to the presence of a non-63 specific stem-loop as well as the specific RBS sequence [30][31][32]. Secondary ...

Aspergillus fumigatus is a ubiquitous environmental mold that causes significant mortality particularly amongst immunocompromised patients. The detection of the Aspergillus-derived carbohydrate galactomannan in patient sera and bronchoalveolar lavage fluid is the major biomarker used to detect A. fumigatus infection in clinical medicine. Despite the clinical relevance of this carbohydrate, we lack a fundamental understanding of how galactomannan is recognized by the innate immune system. Galactomannan is composed of a linear mannan backbone with galactofuranose sidechains and is found both attached to the cell surface of Aspergillus and as a soluble carbohydrate in the extracellular milieu. In this study, we utilized fungal-like particles composed of purified Aspergillus galactomannan to identify a C-type lectin host receptor responsible for binding this fungal carbohydrate. We identified a novel interaction between Aspergillus galactomannan and the C-type lectin receptor Dectin-2. We demonstrate that galactomannan bound to Dectin-2 and induced Dectin-2 dependent signaling including activation of spleen tyrosine kinase and potent TNFα production. Deficiency of Dectin-2 increased neutrophil recruitment to the lungs but was dispensable for survival in a mouse model of pulmonary aspergillosis. Our results identify a novel interaction between galactomannan and Dectin-2 and demonstrate that Dectin-2 is a receptor for Aspergillus galactomannan which leads to a pro-inflammatory response.

17The success of Mycobacterium tuberculosis (Mtb) as a human pathogen is due in part to its 18 ability to survive stress conditions, such as hypoxia or nutrient deprivation, by entering non-19 growing states. In these low-metabolic states, Mtb can tolerate antibiotics and develop 20 genetically encoded antibiotic resistance, making its metabolic adaptation to stress crucial for 21 survival. Numerous bacteria, including Mtb, have been shown to reduce their rates of mRNA 22 degradation under growth limitation and stress. While the existence of this response appears to 23 be conserved across species, the underlying bacterial mRNA stabilization mechanisms remains 24 65 for other bacteria under stress conditions that slow or halt growth, including carbon deprivation, 66 stationary phase, and temperature shock (4-13). However, the mechanisms responsible for global 67 regulation of mRNA stability in prokaryotes have yet to be elucidated. 68 In better studied bacteria such as E. coli and B. subtilis, the major ribonucleases (RNases) 69 involved in mRNA processing and decay are RNase E and RNase Y, respectively. A 70 conventional model for RNA decay in E. coli start with an endonucleolytic cleavage event 71 usually carried by RNase E in AU-rich regions, particularly in mRNA substrates that possess a 5' 72 monophosphate (14-16). The resulting 5' monophosphorylated fragments are rapidly cleaved by 73 RNase E, resulting in shorter fragments that can be fully degraded by exonucleases such as 74 PNPase, RNase II, and RNase R (17, 18). mRNA degradation seems to be coordinated by 75 formation of a complex known as the degradosome. In E. coli, RNase E serves as the scaffold for 76 this multiprotein complex that comprises RNA helicases, the glycolytic enzyme enolase, and 77 PNPase (19-23). Other organisms that encode RNase E form similar degradosomes (24, 25). In 78 organisms where RNase E is not present, RNase Y and/or RNase J seem to assume the scaffold 79 function (26-28). Mycobacteria encode RNase E, but efforts to define the mycobacterial 80 degradosome have produced inconsistent results (29, 30). It is unclear if degradosome 81 reorganization or dissolution contribute to the global regulation of mRNA degradation under 82 stress conditions in any bacteria. Interestingly, the importance of degradosome formation in E. 83 coli varies depending on the carbon sources provided, suggesting specific links between RNase E 84 degradosomes and metabolic capabilities (31). Furthermore, the chaperones DnaK and CsdA can 85 become degradosome components in E. coli under certain stresses (20, 32, 33). 86Global transcript stabilization in stressed bacteria could plausibly result from reduced RNase 87 abundance, reduced RNase activity, and/or reduced accessibility of transcripts to degradation 88 proteins. In E. coli it has been shown that multiple stressors can upregulate RNase R, possibly as 89 a way to overcome ribosome misassembly (34, 35), and that RNase III levels decrease under 90 cold-shock and stationary phase (36). Surprisin...