Transcription-replication interactions reveal bacterial genome regulation.

Organisms determine the transcription rates of thousands of genes through a few modes of regulation that recur across the genome1. In bacteria, the relationship between the regulatory architecture of a gene and its expression is well understood for individual model gene circuits2,3. However, a broader perspective of these dynamics at the genome scale is lacking, in part because bacterial transcriptomics has hitherto captured only a static snapshot of expression averaged across millions of cells4. As a result, the full diversity of gene expression dynamics and their relation to regulatory architecture remains unknown. Here we present a novel genome-wide classification of regulatory modes based on the transcriptional response of each gene to its own replication, which we term the transcription-replication interaction profile (TRIP). Analysing single-bacterium RNA-sequencing data, we found that the response to the universal perturbation of chromosomal replication integrates biological regulatory factors with biophysical molecular events on the chromosome to reveal the local regulatory context of a gene. Whereas the TRIPs of many genes conform to a gene dosage-dependent pattern, others diverge in distinct ways, and this is shaped by factors such as intra-operon position and repression state. By revealing the underlying mechanistic drivers of gene expression heterogeneity, this work provides a quantitative, biophysical framework for modelling replication-dependent expression dynamics.

Candida auris-macrophage cellular interactions and transcriptional response.

The pathogenic yeast Candida auris represents a global threat of the utmost clinical relevance. This emerging fungal species is remarkable in its resistance to commonly used antifungal agents and its persistence in the nosocomial settings. The innate immune system is one the first lines of defense preventing the dissemination of pathogens in the host. C. auris is susceptible to circulating phagocytes, and understanding the molecular details of these interactions may suggest routes to improved therapies. In this work, we examined the interactions of this yeast with macrophages. We found that macrophages avidly phagocytose C. auris; however, intracellular replication is not inhibited, indicating that C. auris resists the killing mechanisms imposed by the phagocyte. Unlike Candida albicans, phagocytosis of C. auris does not induce macrophage lysis. The transcriptional response of C. auris to macrophage phagocytosis is very similar to other members of the CUG clade (C. albicans, C. tropicalis, C. parapsilosis, C. lusitaniae), i.e., downregulation of transcription/translation and upregulation of alternative carbon metabolism pathways, transporters, and induction of oxidative stress response and proteolysis. Gene family expansions are common in this yeast, and we found that many of these genes are induced in response to macrophage co-incubation. Among these, amino acid and oligopeptide transporters, as well as lipases and proteases, are upregulated. Thus, C. auris shares key transcriptional signatures shared with other fungal pathogens and capitalizes on the expansion of gene families coding for potential virulence attributes that allow its survival, persistence, and evasion of the innate immune system.

Veterinary trypanocidal benzoxaboroles are peptidase-activated prodrugs.

Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism.

The Paralogous Transcription Factors Stp1 and Stp2 of Candida albicans Have Distinct Functions in Nutrient Acquisition and Host Interaction.

Nutrient acquisition is a central challenge for all organisms. For the fungal pathogen Candida albicans, utilization of amino acids has been shown to be critical for survival, immune evasion, and escape, while the importance of catabolism of host-derived proteins and peptides in vivo is less well understood. Stp1 and Stp2 are paralogous transcription factors (TFs) regulated by the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing system and have been proposed to have distinct, if uncertain, roles in protein and amino acid utilization. We show here that Stp1 is required for proper utilization of peptides but has no effect on amino acid catabolism. In contrast, Stp2 is critical for utilization of both carbon sources. Commensurate with this observation, we found that Stp1 controls a very limited set of genes, while Stp2 has a much more extensive regulon that is partly dependent on the Ssy1 amino acid sensor (amino acid uptake and catabolism) and partly Ssy1 independent (genes associated with filamentous growth, including the regulators UME6 and SFL2). The ssy1Δ/Δ and stp2Δ/Δ mutants showed reduced fitness in a gastrointestinal (GI) colonization model, yet induced greater damage to epithelial cells and macrophages in a manner that was highly dependent on the growth status of the fungal cells. Surprisingly, the stp1Δ/Δ mutant was better able to colonize the gut but the mutation had no effect on host cell damage. Thus, proper protein and amino acid utilization are both required for normal host interaction and are controlled by an interrelated network that includes Stp1 and Stp2.

Deletion of transketolase triggers a stringent metabolic response in promastigotes and loss of virulence in amastigotes of Leishmania mexicana.

Transketolase (TKT) is part of the non-oxidative branch of the pentose phosphate pathway (PPP). Here we describe the impact of removing this enzyme from the pathogenic protozoan Leishmania mexicana. Whereas the deletion had no obvious effect on cultured promastigote forms of the parasite, the Δtkt cells were not virulent in mice. Δtkt promastigotes were more susceptible to oxidative stress and various leishmanicidal drugs than wild-type, and metabolomics analysis revealed profound changes to metabolism in these cells. In addition to changes consistent with those directly related to the role of TKT in the PPP, central carbon metabolism was substantially decreased, the cells consumed significantly less glucose, flux through glycolysis diminished, and production of the main end products of metabolism was decreased. Only minor changes in RNA abundance from genes encoding enzymes in central carbon metabolism, however, were detected although fructose-1,6-bisphosphate aldolase activity was decreased two-fold in the knock-out cell line. We also showed that the dual localisation of TKT between cytosol and glycosomes is determined by the C-terminus of the enzyme and by engineering different variants of the enzyme we could alter its sub-cellular localisation. However, no effect on the overall flux of glucose was noted irrespective of whether the enzyme was found uniquely in either compartment, or in both.

Transcription-replication interactions reveal principles of bacterial genome regulation.

Organisms determine the transcription rates of thousands of genes through a few modes of regulation that recur across the genome1. These modes interact with a changing cellular environment to yield highly dynamic expression patterns2. In bacteria, the relationship between a gene's regulatory architecture and its expression is well understood for individual model gene circuits3,4. However, a broader perspective of these dynamics at the genome-scale is lacking, in part because bacterial transcriptomics have hitherto captured only a static snapshot of expression averaged across millions of cells5. As a result, the full diversity of gene expression dynamics and their relation to regulatory architecture remains unknown. Here we present a novel genome-wide classification of regulatory modes based on each gene's transcriptional response to its own replication, which we term the Transcription-Replication Interaction Profile (TRIP). We found that the response to the universal perturbation of chromosomal replication integrates biological regulatory factors with biophysical molecular events on the chromosome to reveal a gene's local regulatory context. While the TRIPs of many genes conform to a gene dosage-dependent pattern, others diverge in distinct ways, including altered timing or amplitude of expression, and this is shaped by factors such as intra-operon position, repression state, or presence on mobile genetic elements. Our transcriptome analysis also simultaneously captures global properties, such as the rates of replication and transcription, as well as the nestedness of replication patterns. This work challenges previous notions of the drivers of expression heterogeneity within a population of cells, and unearths a previously unseen world of gene transcription dynamics.

The tempo and mode of gene regulatory programs during bacterial infection.

Innate immune recognition of bacterial pathogens is a key determinant of the ensuing systemic response, and host or pathogen heterogeneity in this early interaction can impact the course of infection. To gain insight into host response heterogeneity, we investigate macrophage inflammatory dynamics using primary human macrophages infected with Group B Streptococcus. Transcriptomic analysis reveals discrete cellular states within responding macrophages, one of which consists of four sub-states, reflecting inflammatory activation. Infection with six additional bacterial species-Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Yersinia pseudotuberculosis, Shigella flexneri, and Salmonella enterica-recapitulates these states, though at different frequencies. We show that modulating the duration of infection and the presence of a toxin impacts inflammatory trajectory dynamics. We provide evidence for this trajectory in infected macrophages in an in vivo model of Staphylococcus aureus infection. Our cell-state analysis defines a framework for understanding inflammatory activation dynamics in response to bacterial infection.

Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism and oxidative stress response.

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each resistant line, the predominant wild-type sterol, ergosta-5,7,24-trienol, was replaced by other sterol intermediates. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter gene was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for their ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several AmB resistant lines showed reduced virulence, at least two lines displayed heightened virulence in mice whilst retaining their resistance phenotype, emphasising the risks of resistance emerging to this critical drug.

Interactions of Both Pathogenic and Nonpathogenic CUG Clade Candida Species with Macrophages Share a Conserved Transcriptional Landscape.

Candida species are a leading cause of opportunistic, hospital-associated bloodstream infections with high mortality rates, typically in immunocompromised patients. Several species, including Candida albicans, the most prevalent cause of infection, belong to the monophyletic CUG clade of yeasts. Innate immune cells such as macrophages are crucial for controlling infection, and C. albicans responds to phagocytosis by a coordinated induction of pathways involved in catabolism of nonglucose carbon sources, termed alternative carbon metabolism, which together are essential for virulence. However, the interactions of other CUG clade species with macrophages have not been characterized. Here, we analyzed transcriptional responses to macrophage phagocytosis by six Candida species across a range of virulence and clinical importance. We define a core induced response common to pathogenic and nonpathogenic species alike, heavily weighted to alternative carbon metabolism. One prominent pathogen, Candida parapsilosis, showed species-specific expansion of phagocytosis-responsive genes, particularly metabolite transporters. C. albicans and Candida tropicalis, the other prominent pathogens, also had species-specific responses, but these were largely comprised of functionally uncharacterized genes. Transcriptional analysis of macrophages also demonstrated highly correlated proinflammatory transcriptional responses to different Candida species that were largely independent of fungal viability, suggesting that this response is driven by recognition of conserved cell wall components. This study significantly broadens our understanding of host interactions in CUG clade species, demonstrating that although metabolic plasticity is crucial for virulence in Candida, it alone is not sufficient to confer pathogenicity. Instead, we identify sets of mostly uncharacterized genes that may explain the evolution of pathogenicity. IMPORTANCE Candidiasis is a major fungal infection by Candida species, causing life-threatening invasive disease in immunocompromised patients. C. albicans, which is adapted to commensalism of human mucosae, is the most common cause. While several other species cause infection, most are less prevalent or less virulent. As innate immune cells are the primary defense against Candida infection, we compared the transcriptional responses of C. albicans and related species to phagocytosis by macrophages, to understand the basis of variation in pathogenesis. This response, including the metabolic remodeling required for virulence in C. albicans, was strikingly conserved across the virulence spectrum. Macrophage responses to different species were also highly similar. This study indicates that important elements of host-pathogen interactions in C. albicans are not driven by adaptation to the mammalian host and improves our understanding of pathogenicity in opportunistic fungal species that are understudied but collectively impose a significant threat of their own.

Untargeted metabolomics to understand the basis of phenotypic differences in amphotericin B-resistant Leishmania parasites.

Background: Protozoan Leishmania parasites are responsible for a range of clinical infections that represent a substantial challenge for global health. Amphotericin B (AmB) is increasingly used to treat Leishmania infection, so understanding the potential for resistance to this drug is an important priority. Previously we described four independently-derived AmB-resistant L. mexicana lines that exhibited resistance-associated genetic lesions resulting in altered sterol content. However, substantial phenotypic variation between these lines, including differences in virulence attributes, were not fully explained by these changes. Methods: To identify alterations in cellular metabolism potentially related to phenotypic differences between wild-type and AmB-resistant lines, we extracted metabolites and performed untargeted metabolomics by liquid chromatography-mass spectrometry. Results: We observed substantial differences in metabolite abundance between lines, arising in an apparently stochastic manner. Concerted remodeling of central carbon metabolism was not observed; however, in three lines, decreased abundance of several oligohexoses was observed. Given that the oligomannose mannogen is an important virulence factor in Leishmania, this could relate to loss of virulence in these lines. Increased abundance of the reduced forms of the oxidative stress-protective thiols trypanothione and glutathione was also observed in multiple lines. Conclusions: This dataset will provide a useful resource for understanding the molecular basis of drug resistance in Leishmania, and suggests a role for metabolic changes separate from the primary mechanism of drug resistance in determining the phenotypic profile of parasite lines subjected to experimental selection of resistance.

Genomic instability at the locus of sterol C24-methyltransferase promotes amphotericin B resistance in Leishmania parasites.

Amphotericin B is an increasingly important tool in efforts to reduce the global disease burden posed by Leishmania parasites. With few other chemotherapeutic options available for the treatment of leishmaniasis, the potential for emergent resistance to this drug is a considerable threat. Here we characterised four novel amphotericin B-resistant Leishmania mexicana lines. All lines exhibited altered sterol biosynthesis, and hypersensitivity to pentamidine. Whole genome sequencing demonstrated resistance-associated mutation of the sterol biosynthesis gene sterol C5-desaturase in one line. However, in three out of four lines, RNA-seq revealed loss of expression of sterol C24-methyltransferase (SMT) responsible for drug resistance and altered sterol biosynthesis. Additional loss of the miltefosine transporter was associated with one of those lines. SMT is encoded by two tandem gene copies, which we found to have very different expression levels. In all cases, reduced overall expression was associated with loss of the 3' untranslated region of the dominant gene copy, resulting from structural variations at this locus. Local regions of sequence homology, between the gene copies themselves, and also due to the presence of SIDER1 retrotransposon elements that promote multi-gene amplification, correlate to these structural variations. Moreover, in at least one case loss of SMT expression was not associated with loss of virulence in primary macrophages or in vivo. Whilst such repeat sequence-mediated instability is known in Leishmania genomes, its presence associated with resistance to a major antileishmanial drug, with no evidence of associated fitness costs, is a significant concern.

Metabolomic profiling of macrophages determines the discrete metabolomic signature and metabolomic interactome triggered by polarising immune stimuli.

Priming and activating immune stimuli have profound effects on macrophages, however, studies generally evaluate stimuli in isolation rather than in combination. In this study we have investigated the effects of pro-inflammatory and anti-inflammatory stimuli either alone or in combination on macrophage metabolism. These stimuli include host factors such as IFNγ and ovalbumin-immunoglobulin immune complexes, or pathogen factors such as LPS. Untargeted LC-MS based metabolomics provided an in-depth profile of the macrophage metabolome, and revealed specific changes in metabolite abundance upon either individual stimuli or combined stimuli. Here, by factoring in an interaction term in the linear model, we define the metabolome interactome. This approach allowed us to determine whether stimuli interact in a synergistic or antagonistic manner. In conclusion this study demonstrates a robust approach to interrogate immune-metabolism, especially systems that model host-pathogen interactions.

Drug resistance and treatment failure in leishmaniasis: A 21st century challenge.

Reevaluation of treatment guidelines for Old and New World leishmaniasis is urgently needed on a global basis because treatment failure is an increasing problem. Drug resistance is a fundamental determinant of treatment failure, although other factors also contribute to this phenomenon, including the global HIV/AIDS epidemic with its accompanying impact on the immune system. Pentavalent antimonials have been used successfully worldwide for the treatment of leishmaniasis since the first half of the 20th century, but the last 10 to 20 years have witnessed an increase in clinical resistance, e.g., in North Bihar in India. In this review, we discuss the meaning of "resistance" related to leishmaniasis and discuss its molecular epidemiology, particularly for Leishmania donovani that causes visceral leishmaniasis. We also discuss how resistance can affect drug combination therapies. Molecular mechanisms known to contribute to resistance to antimonials, amphotericin B, and miltefosine are also outlined.

Sterol 14α-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana.

Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administration of the drug in its liposomal formulation as a single injection is being promoted in a campaign to bring the leishmaniases under control. Understanding the risks and mechanisms of resistance is therefore of great importance. Here we select amphotericin B-resistant Leishmania mexicana parasites with relative ease. Metabolomic analysis demonstrated that ergosterol, the sterol known to bind the drug, is prevalent in wild-type cells, but diminished in the resistant line, where alternative sterols become prevalent. This indicates that the resistance phenotype is related to loss of drug binding. Comparing sequences of the parasites' genomes revealed a plethora of single nucleotide polymorphisms that distinguish wild-type and resistant cells, but only one of these was found to be homozygous and associated with a gene encoding an enzyme in the sterol biosynthetic pathway, sterol 14α-demethylase (CYP51). The mutation, N176I, is found outside of the enzyme's active site, consistent with the fact that the resistant line continues to produce the enzyme's product. Expression of wild-type sterol 14α-demethylase in the resistant cells caused reversion to drug sensitivity and a restoration of ergosterol synthesis, showing that the mutation is indeed responsible for resistance. The amphotericin B resistant parasites become hypersensitive to pentamidine and also agents that induce oxidative stress. This work reveals the power of combining polyomics approaches, to discover the mechanism underlying drug resistance as well as offering novel insights into the selection of resistance to amphotericin B itself.