Small-Molecule Antibiotic Drug Development: Need and Challenges.

The need for new antibiotics is urgent. Antimicrobial resistance is rising, although currently, many more people die from drug-sensitive bacterial infections. The continued evolution of drug resistance is inevitable, fueled by pathogen population size and exposure to antibiotics. Additionally, opportunistic pathogens will always pose a threat to vulnerable patients whose immune systems cannot efficiently fight them even if they are sensitive to available antibiotics, according to clinical microbiology tests. These problems are intertwined and will worsen as human populations age, increase in density, and experience disruptions such as war, extreme weather events, or declines in standard of living. The development of appropriate drugs to treat all the world's bacterial infections should be a priority, and future success will likely require combinations of multiple approaches. However, the highest burden of bacterial infection is in Low- and Middle-Income Countries, where limited medical infrastructure is a major challenge. For effectively managing infections in these contexts, small-molecule-based treatments offer significant advantages. Unfortunately, support for ongoing small-molecule antibiotic discovery has recently suffered from significant challenges related both to the scientific difficulties in treating bacterial infections and to market barriers. Nevertheless, small-molecule antibiotics remain essential and irreplaceable tools for fighting infections, and efforts to develop novel and improved versions deserve ongoing investment. Here, we first describe the global historical context of antibiotic treatment and then highlight some of the challenges surrounding small-molecule development and potential solutions. Many of these challenges are likely to be common to all modalities of antibacterial treatment and should be addressed directly.

An anti-biofilm cyclic peptide targets a secreted aminopeptidase from P. aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes serious illness, especially in immunocompromised individuals. P. aeruginosa forms biofilms that contribute to growth and persistence in a wide range of environments. Here we investigated the aminopeptidase, P. aeruginosa aminopeptidase (PaAP) from P. aeruginosa, which is highly abundant in the biofilm matrix. PaAP is associated with biofilm development and contributes to nutrient recycling. We confirmed that post-translational processing was required for activation and PaAP is a promiscuous aminopeptidase acting on unstructured regions of peptides and proteins. Crystal structures of wild-type enzymes and variants revealed the mechanism of autoinhibition, whereby the C-terminal propeptide locks the protease-associated domain and the catalytic peptidase domain into a self-inhibited conformation. Inspired by this, we designed a highly potent small cyclic-peptide inhibitor that recapitulates the deleterious phenotype observed with a PaAP deletion variant in biofilm assays and present a path toward targeting secreted proteins in a biofilm context.

Bacterial transcription during growth arrest.

Bacteria in most natural environments spend substantial periods of time limited for essential nutrients and not actively dividing. While transcriptional activity under these conditions is substantially reduced compared to that occurring during active growth, observations from diverse organisms and experimental approaches have shown that new transcription still occurs and is important for survival. Much of our understanding of transcription regulation has come from measuring transcripts in exponentially growing cells, or from in vitro experiments focused on transcription from highly active promoters by the housekeeping RNA polymerase holoenzyme. The fact that transcription during growth arrest occurs at low levels and is highly heterogeneous has posed challenges for its study. However, new methods of measuring low levels of gene expression activity, even in single cells, offer exciting opportunities for directly investigating transcriptional activity and its regulation during growth arrest. Furthermore, much of the rich structural and biochemical data from decades of work on the bacterial transcriptional machinery is also relevant to growth arrest. In this review, the physiological changes likely affecting transcription during growth arrest are first considered. Next, possible adaptations to help facilitate ongoing transcription during growth arrest are discussed. Finally, new insights from several recently published datasets investigating mRNA transcripts in single bacterial cells at various growth phases will be explored. Keywords: Growth arrest, stationary phase, RNA polymerase, nucleoid condensation, population heterogeneity.

Diversity in Starvation Survival Strategies and Outcomes among Heterotrophic Proteobacteria.

Heterotrophic Proteobacteria are versatile opportunists that have been extensively studied as model organisms in the laboratory, as both pathogens and beneficial symbionts of plants and animals, and as ubiquitous organisms found free-living in many environments. Succeeding in these niches requires an ability to persist for potentially long periods of time in growth-arrested states when essential nutrients become limiting. The tendency of these bacteria to grow in dense biofilm communities frequently leads to the development of steep nutrient gradients and deprivation of interior cells even when the environment is nutrient rich. Surviving within host environments also likely requires tolerating growth arrest due to the host limiting access to nutrients and transitioning between hosts may require a period of survival in a nutrient-poor environment. Interventions to maximise plant-beneficial activities and minimise infections by bacteria will require a better understanding of metabolic and regulatory networks that contribute to starvation survival, and how these networks function in diverse organisms. Here we focus on carbon starvation as a growth-arresting condition that limits availability not only of substrates for biosynthesis but also of energy for ongoing maintenance of the electrochemical gradient across the cell envelope and cellular integrity. We first review models for studying bacterial starvation and known strategies that contribute to starvation survival. We then present the results of a survey of carbon starvation survival strategies and outcomes in ten bacterial strains, including representatives from the orders Enterobacterales and Pseudomonadales (both Gammaproteobacteria) and Burkholderiales (Betaproteobacteria). Finally, we examine differences in gene content between the highest and lowest survivors to identify metabolic and regulatory adaptations that may contribute to differences in starvation survival.

Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics.

Bacterial opportunistic human pathogens frequently exhibit intrinsic antibiotic tolerance and resistance, resulting in infections that can be nearly impossible to eradicate. We asked whether this recalcitrance could be driven by these organisms' evolutionary history as environmental microbes that engage in chemical warfare. Using Pseudomonas aeruginosa as a model, we demonstrate that the self-produced antibiotic pyocyanin (PYO) activates defenses that confer collateral tolerance specifically to structurally similar synthetic clinical antibiotics. Non-PYO-producing opportunistic pathogens, such as members of the Burkholderia cepacia complex, likewise display elevated antibiotic tolerance when cocultured with PYO-producing strains. Furthermore, by widening the population bottleneck that occurs during antibiotic selection and promoting the establishment of a more diverse range of mutant lineages, PYO increases apparent rates of mutation to antibiotic resistance to a degree that can rival clinically relevant hypermutator strains. Together, these results reveal an overlooked mechanism by which opportunistic pathogens that produce natural toxins can dramatically modulate the efficacy of clinical antibiotics and the evolution of antibiotic resistance, both for themselves and other members of clinically relevant polymicrobial communities.

Regulation of protein biosynthetic activity during growth arrest.

Heterotrophic bacteria grow and divide rapidly when resources are abundant. Yet resources are finite, and environments fluctuate, so bacteria need strategies to survive when nutrients become scarce. In fact, many bacteria spend most of their time in such conditions of nutrient limitation, and hence they need to optimise gene regulation and protein biosynthesis during growth arrest. An optimal strategy in these conditions must mitigate the challenges and risks of making new proteins, while the cell is severely limited for energy and substrates. Recently, ribosome abundance and activity were measured in these conditions, revealing very low amounts of new protein synthesis, which is nevertheless vital for survival. The underlying mechanisms are only now starting to be explored. Improving our understanding of the regulation of protein production during bacterial growth arrest could have important implications for a wide range of challenges, including the identification of new targets for antibiotic development.

Aggregation of Nontuberculous Mycobacteria Is Regulated by Carbon-Nitrogen Balance.

Nontuberculous mycobacteria (NTM) are emerging opportunistic pathogens that colonize household water systems and cause chronic lung infections in susceptible patients. The ability of NTM to form surface-attached biofilms in the nonhost environment and corded aggregates in vivo is important to their ability to persist in both contexts. Underlying the development of these multicellular structures is the capacity of mycobacterial cells to adhere to one another. Unlike most other bacteria, NTM spontaneously and constitutively aggregate in vitro, hindering our ability to understand the transition between planktonic and aggregated cells. While culturing a model NTM, Mycobacterium smegmatis, in rich medium, we fortuitously discovered that planktonic cells accumulate after ∼3 days of growth. By providing selective pressure for bacteria that disperse earlier, we isolated a strain with two mutations in the oligopeptide permease operon (opp). A mutant lacking the opp operon (Δopp) disperses earlier than wild type (WT) due to a defect in nutrient uptake. Experiments with WT M. smegmatis revealed that growth as aggregates is favored when carbon is replete, but under conditions of low available carbon relative to available nitrogen, M. smegmatis grows as planktonic cells. By adjusting carbon and nitrogen sources in defined medium, we tuned the cellular C/N ratio such that M. smegmatis grows either as aggregates or as planktonic cells. C/N-mediated aggregation regulation is widespread among NTM with the possible exception of rough-colony Mycobacterium abscessus isolates. Altogether, we show that NTM aggregation is a controlled process that is governed by the relative availability of carbon and nitrogen for metabolism.IMPORTANCE Free-living bacteria can assemble into multicellular structures called biofilms. Biofilms help bacteria tolerate multiple stresses, including antibiotics and the host immune system. Nontuberculous mycobacteria are a group of emerging opportunistic pathogens that utilize biofilms to adhere to household plumbing and showerheads and to avoid phagocytosis by host immune cells. Typically, bacteria regulate biofilm formation by controlling expression of adhesive structures to attach to surfaces and other bacterial cells. Mycobacteria harbor a unique cell wall built chiefly of long-chain mycolic acids that confers hydrophobicity and has been thought to cause constitutive aggregation in liquid media. Here we show that aggregation is instead a regulated process dictated by the balance of available carbon and nitrogen. Understanding that mycobacteria utilize metabolic cues to regulate the transition between planktonic and aggregated cells reveals an inroad to controlling biofilm formation through targeted therapeutics.

The dormancy-specific regulator, SutA, is intrinsically disordered and modulates transcription initiation in Pseudomonas aeruginosa.

Though most bacteria in nature are nutritionally limited and grow slowly, our understanding of core processes like transcription comes largely from studies in model organisms doubling rapidly. We previously identified a small protein of unknown function, SutA, in a screen of proteins synthesized in Pseudomonas aeruginosa during dormancy. SutA binds RNA polymerase (RNAP), causing widespread changes in gene expression, including upregulation of the ribosomal RNA genes. Here, using biochemical and structural methods, we examine how SutA interacts with RNAP and the functional consequences of these interactions. We show that SutA comprises a central α-helix with unstructured N- and C-terminal tails, and binds to the ß1 domain of RNAP. It activates transcription from the rrn promoter by both the housekeeping sigma factor holoenzyme (Eσ70 ) and the stress sigma factor holoenzyme (EσS ) in vitro, but has a greater impact on EσS . In both cases, SutA appears to affect intermediates in the open complex formation and its N-terminal tail is required for activation. The small magnitudes of in vitro effects are consistent with a role in maintaining activity required for homeostasis during dormancy. Our results add SutA to a growing list of transcription regulators that use their intrinsically disordered regions to remodel transcription complexes.

Identification of Fitness Determinants during Energy-Limited Growth Arrest in Pseudomonas aeruginosa.

Microbial growth arrest can be triggered by diverse factors, one of which is energy limitation due to scarcity of electron donors or acceptors. Genes that govern fitness during energy-limited growth arrest and the extent to which they overlap between different types of energy limitation are poorly defined. In this study, we exploited the fact that Pseudomonas aeruginosa can remain viable over several weeks when limited for organic carbon (pyruvate) as an electron donor or oxygen as an electron acceptor. ATP values were reduced under both types of limitation, yet more severely in the absence of oxygen. Using transposon-insertion sequencing (Tn-seq), we identified fitness determinants in these two energy-limited states. Multiple genes encoding general functions like transcriptional regulation and energy generation were required for fitness during carbon or oxygen limitation, yet many specific genes, and thus specific activities, differed in their relevance between these states. For instance, the global regulator RpoS was required during both types of energy limitation, while other global regulators such as DksA and LasR were required only during carbon or oxygen limitation, respectively. Similarly, certain ribosomal and tRNA modifications were specifically required during oxygen limitation. We validated fitness defects during energy limitation using independently generated mutants of genes detected in our screen. Mutants in distinct functional categories exhibited different fitness dynamics: regulatory genes generally manifested a phenotype early, whereas genes involved in cell wall metabolism were required later. Together, these results provide a new window into how P. aeruginosa survives growth arrest.IMPORTANCE Growth-arrested bacteria are ubiquitous in nature and disease yet understudied at the molecular level. For example, growth-arrested cells constitute a major subpopulation of mature biofilms, serving as an antibiotic-tolerant reservoir in chronic infections. Identification of the genes required for survival of growth arrest (encompassing entry, maintenance, and exit) is an important first step toward understanding the physiology of bacteria in this state. Using Tn-seq, we identified and validated genes required for fitness of Pseudomonas aeruginosa when energy limited for organic carbon or oxygen, which represent two common causes of growth arrest for P. aeruginosa in diverse habitats. This unbiased, genome-wide survey is the first to reveal essential activities for a pathogen experiencing different types of energy limitation, finding both shared and divergent activities that are relevant at different survival stages. Future efforts can now be directed toward understanding how the biomolecules responsible for these activities contribute to fitness under these conditions.

The Evolutionarily-conserved Polyadenosine RNA Binding Protein, Nab2, Cooperates with Splicing Machinery to Regulate the Fate of pre-mRNA.

Numerous RNA binding proteins are deposited onto an mRNA transcript to modulate post-transcriptional processing events ensuring proper mRNA maturation. Defining the interplay between RNA binding proteins that couple mRNA biogenesis events is crucial for understanding how gene expression is regulated. To explore how RNA binding proteins control mRNA processing, we investigated a role for the evolutionarily conserved polyadenosine RNA binding protein, Nab2, in mRNA maturation within the nucleus. This work reveals that nab2 mutant cells accumulate intron-containing pre-mRNA in vivo We extend this analysis to identify genetic interactions between mutant alleles of nab2 and genes encoding the splicing factor, MUD2, and the RNA exosome, RRP6, with in vivo consequences of altered pre-mRNA splicing and poly(A) tail length control. As further evidence linking Nab2 proteins to splicing, an unbiased proteomic analysis of vertebrate Nab2, ZC3H14, identifies physical interactions with numerous components of the spliceosome. We validated the interaction between ZC3H14 and U2AF2/U2AF65 Taking all the findings into consideration, we present a model where Nab2/ZC3H14 interacts with spliceosome components to allow proper coupling of splicing with subsequent mRNA processing steps contributing to a kinetic proofreading step that allows properly processed mRNA to exit the nucleus and escape Rrp6-dependent degradation.

The physiology of growth arrest: uniting molecular and environmental microbiology.

Most bacteria spend the majority of their time in prolonged states of very low metabolic activity and little or no growth, in which electron donors, electron acceptors and/or nutrients are limited, but cells are poised to undergo rapid division cycles when resources become available. These non-growing states are far less studied than other growth states, which leaves many questions regarding basic bacterial physiology unanswered. In this Review, we discuss findings from a small but diverse set of systems that have been used to investigate how growth-arrested bacteria adjust metabolism, regulate transcription and translation, and maintain their chromosomes. We highlight major questions that remain to be addressed, and suggest that progress in answering them will be aided by recent methodological advances and by dialectic between environmental and molecular microbiology perspectives.

SutA is a bacterial transcription factor expressed during slow growth in Pseudomonas aeruginosa.

Microbial quiescence and slow growth are ubiquitous physiological states, but their study is complicated by low levels of metabolic activity. To address this issue, we used a time-selective proteome-labeling method [bioorthogonal noncanonical amino acid tagging (BONCAT)] to identify proteins synthesized preferentially, but at extremely low rates, under anaerobic survival conditions by the opportunistic pathogen Pseudomonas aeruginosa. One of these proteins is a transcriptional regulator that has no homology to any characterized protein domains and is posttranscriptionally up-regulated during survival and slow growth. This small, acidic protein associates with RNA polymerase, and chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing suggests that the protein associates with genomic DNA through this interaction. ChIP signal is found both in promoter regions and throughout the coding sequences of many genes and is particularly enriched at ribosomal protein genes and in the promoter regions of rRNA genes. Deletion of the gene encoding this protein affects expression of these and many other genes and impacts biofilm formation, secondary metabolite production, and fitness in fluctuating conditions. On the basis of these observations, we have designated the protein SutA (survival under transitions A).

Enzymatic Degradation of Phenazines Can Generate Energy and Protect Sensitive Organisms from Toxicity.

UNLABELLED: Diverse bacteria, including several Pseudomonas species, produce a class of redox-active metabolites called phenazines that impact different cell types in nature and disease. Phenazines can affect microbial communities in both positive and negative ways, where their presence is correlated with decreased species richness and diversity. However, little is known about how the concentration of phenazines is modulated in situ and what this may mean for the fitness of members of the community. Through culturing of phenazine-degrading mycobacteria, genome sequencing, comparative genomics, and molecular analysis, we identified several conserved genes that are important for the degradation of three Pseudomonas-derived phenazines: phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), and pyocyanin (PYO). PCA can be used as the sole carbon source for growth by these organisms. Deletion of several genes in Mycobacterium fortuitum abolishes the degradation phenotype, and expression of two genes in a heterologous host confers the ability to degrade PCN and PYO. In cocultures with phenazine producers, phenazine degraders alter the abundance of different phenazine types. Not only does degradation support mycobacterial catabolism, but also it provides protection to bacteria that would otherwise be inhibited by the toxicity of PYO. Collectively, these results serve as a reminder that microbial metabolites can be actively modified and degraded and that these turnover processes must be considered when the fate and impact of such compounds in any environment are being assessed. IMPORTANCE: Phenazine production by Pseudomonas spp. can shape microbial communities in a variety of environments ranging from the cystic fibrosis lung to the rhizosphere of dryland crops. For example, in the rhizosphere, phenazines can protect plants from infection by pathogenic fungi. The redox activity of phenazines underpins their antibiotic activity, as well as providing pseudomonads with important physiological benefits. Our discovery that soil mycobacteria can catabolize phenazines and thereby protect other organisms against phenazine toxicity suggests that phenazine degradation may influence turnover in situ. The identification of genes involved in the degradation of phenazines opens the door to monitoring turnover in diverse environments, an essential process to consider when one is attempting to understand or control communities influenced by phenazines.

The upper respiratory tract as a microbial source for pulmonary infections in cystic fibrosis. Parallels from island biogeography.

A continuously mixed series of microbial communities inhabits various points of the respiratory tract, with community composition determined by distance from colonization sources, colonization rates, and extinction rates. Ecology and evolution theory developed in the context of biogeography is relevant to clinical microbiology and could reframe the interpretation of recent studies comparing communities from lung explant samples, sputum samples, and oropharyngeal swabs. We propose an island biogeography model of the microbial communities inhabiting different niches in human airways. Island biogeography as applied to communities separated by time and space is a useful parallel for exploring microbial colonization of healthy and diseased lungs, with the potential to inform our understanding of microbial community dynamics and the relevance of microbes detected in different sample types. In this perspective, we focus on the intermixed microbial communities inhabiting different regions of the airways of patients with cystic fibrosis.

Yeast-gene replacement using PCR products.

It is often useful to replace a small region of the yeast genome containing a gene of interest with a selectable marker. The selectable marker allows for easy identification of yeast cells that have successfully carried out the gene replacement, and functional consequences of the loss of that gene can then be assessed. The same technique can also be used for removing noncoding portions of the genome that may be of interest, such as promoters or 3' or 5' UTRs, and for introducing tags on the N- or C-termini of proteins (alternatively, see a marker-free yeast gene replacement method on Gene Knockouts, in vivo site-directed mutagenesis and Other Modifications Using the Delitto Perfetto System in Saccharomyces cerevisiae).

Colony PCR.

Colony PCR is a method for rapidly screening colonies of yeast or bacteria that have grown up on selective media following a transformation step, to verify that the desired genetic construct is present, or to amplify a portion of the construct.

Chemical transformation of yeast.

Transformation of chemically competent yeast cells is a method for introducing exogenous DNA into living cells. Typically, the DNA is either a plasmid carrying an autonomous replication sequence that allows for propagation or a linear piece of DNA to be integrated into the genome. The DNA usually also carries a marker that allows for selection of successfully transformed cells by plating on the appropriate selective media.

Diverse environmental stresses elicit distinct responses at the level of pre-mRNA processing in yeast.

Gene expression in eukaryotic cells is profoundly influenced by the post-transcriptional processing of mRNAs, including the splicing of introns in the nucleus and both nuclear and cytoplasmic degradation pathways. These processes have the potential to affect both the steady-state levels and the kinetics of changes to levels of intron-containing transcripts. Here we report the use of a splicing isoform-specific microarray platform to investigate the effects of diverse stress conditions on pre-mRNA processing. Interestingly, we find that diverse stresses cause distinct patterns of changes at this level. The responses we observed are most dramatic for the RPGs and can be categorized into three major classes. The first is characterized by accumulation of RPG pre-mRNA and is seen in multiple types of amino acid starvation regimes; the magnitude of splicing inhibition correlates with the severity of the stress. The second class is characterized by a rapid decrease in both pre- and mature RPG mRNA and is seen in many stresses that inactivate the TORC1 kinase complex. These decreases depend on nuclear turnover of the intron-containing pre-RNAs. The third class is characterized by a decrease in RPG pre-mRNA, with only a modest reduction in the mature species; this response is observed in hyperosmotic and cation-toxic stresses. We show that casein kinase 2 (CK2) makes important contributions to the changes in pre-mRNA processing, particularly for the first two classes of stress responses. In total, our data suggest that complex post-transcriptional programs cooperate to fine-tune expression of intron-containing transcripts in budding yeast.