Genetic and functional enrichments associated with Enterococcus faecalis isolated from the urinary tract.

IMPORTANCE: Urinary tract infection (UTI) is a global health issue that imposes a substantial burden on healthcare systems. Women are disproportionately affected by UTI, with >60% of women experiencing at least one UTI in their lifetime. UTIs can recur, particularly in postmenopausal women, leading to diminished quality of life and potentially life-threatening complications. Understanding how pathogens colonize and survive in the urinary tract is necessary to identify new therapeutic targets that are urgently needed due to rising rates of antimicrobial resistance. How Enterococcus faecalis, a bacterium commonly associated with UTI, adapts to the urinary tract remains understudied. Here, we generated a collection of high-quality closed genome assemblies of clinical urinary E. faecalis isolated from the urine of postmenopausal women that we used alongside detailed clinical metadata to perform a robust comparative genomic investigation of genetic factors that may be involved in E. faecalis survival in the urinary tract.

Functional and genetic adaptations contributing to Enterococcus faecalis persistence in the female urinary tract.

Enterococcus faecalis is the leading Gram-positive bacterial species implicated in urinary tract infection (UTI). An opportunistic pathogen, E. faecalis is a commensal of the human gastrointestinal tract (GIT) and its presence in the GIT is a predisposing factor for UTI. The mechanisms by which E. faecalis colonizes and survives in the urinary tract (UT) are poorly understood, especially in uncomplicated or recurrent UTI. The UT is distinct from the GIT and is characterized by a sparse nutrient landscape and unique environmental stressors. In this study, we isolated and sequenced a collection of 37 clinical E. faecalis strains from the urine of primarily postmenopausal women. We generated 33 closed genome assemblies and four highly contiguous draft assemblies and conducted a comparative genomics to identify genetic features enriched in urinary E. faecalis with respect to E. faecalis isolated from the human GIT and blood. Phylogenetic analysis revealed high diversity among urinary strains and a closer relatedness between urine and gut isolates than blood isolates. Plasmid replicon (rep) typing further underscored possible UT-GIT interconnection identifying nine shared rep types between urine and gut E. faecalis . Both genotypic and phenotypic analysis of antimicrobial resistance among urinary E. faecalis revealed infrequent resistance to front-line UTI antibiotics nitrofurantoin and fluoroquinolones and no vancomycin resistance. Finally, we identified 19 candidate genes enriched among urinary strains that may play a role in adaptation to the UT. These genes are involved in the core processes of sugar transport, cobalamin import, glucose metabolism, and post-transcriptional regulation of gene expression. IMPORTANCE: Urinary tract infection (UTI) is a global health issue that imposes substantial burden on healthcare systems. Women are disproportionately affected by UTI with >60% of women experiencing at least one UTI in their lifetime. UTIs can recur, particularly in postmenopausal women, leading to diminished quality of life and potentially life-threatening complications. Understanding how pathogens colonize and survive in the urinary tract is necessary to identify new therapeutic targets that are urgently needed due to rising rates of antimicrobial resistance. How Enterococcus faecalis , a bacterium commonly associated with UTI, adapts to the urinary tract remains understudied. Here, we generated a collection of high-quality closed genome assemblies of clinical urinary E. faecalis isolated from the urine of postmenopausal women that we used alongside detailed clinical metadata to perform a robust comparative genomic investigation of genetic factors that may mediate urinary E. faecalis adaptation to the female urinary tract.

Structure-Aware Mycobacterium tuberculosis Functional Annotation Uncloaks Resistance, Metabolic, and Virulence Genes.

Accurate and timely functional genome annotation is essential for translating basic pathogen research into clinically impactful advances. Here, through literature curation and structure-function inference, we systematically update the functional genome annotation of Mycobacterium tuberculosis virulent type strain H37Rv. First, we systematically curated annotations for 589 genes from 662 publications, including 282 gene products absent from leading databases. Second, we modeled 1,711 underannotated proteins and developed a semiautomated pipeline that captured shared function between 400 protein models and structural matches of known function on Protein Data Bank, including drug efflux proteins, metabolic enzymes, and virulence factors. In aggregate, these structure- and literature-derived annotations update 940/1,725 underannotated H37Rv genes and generate hundreds of functional hypotheses. Retrospectively applying the annotation to a recent whole-genome transposon mutant screen provided missing function for 48% (13/27) of underannotated genes altering antibiotic efficacy and 33% (23/69) required for persistence during mouse tuberculosis (TB) infection. Prospective application of the protein models enabled us to functionally interpret novel laboratory generated pyrazinamide (PZA)-resistant mutants of unknown function, which implicated the emerging coenzyme A depletion model of PZA action in the mutants' PZA resistance. Our findings demonstrate the functional insight gained by integrating structural modeling and systematic literature curation, even for widely studied microorganisms. Functional annotations and protein structure models are available at https://tuberculosis.sdsu.edu/H37Rv in human- and machine-readable formats. IMPORTANCE Mycobacterium tuberculosis, the primary causative agent of tuberculosis, kills more humans than any other infectious bacterium. Yet 40% of its genome is functionally uncharacterized, leaving much about the genetic basis of its resistance to antibiotics, capacity to withstand host immunity, and basic metabolism yet undiscovered. Irregular literature curation for functional annotation contributes to this gap. We systematically curated functions from literature and structural similarity for over half of poorly characterized genes, expanding the functionally annotated Mycobacterium tuberculosis proteome. Applying this updated annotation to recent in vivo functional screens added functional information to dozens of clinically pertinent proteins described as having unknown function. Integrating the annotations with a prospective functional screen identified new mutants resistant to a first-line TB drug, supporting an emerging hypothesis for its mode of action. These improvements in functional interpretation of clinically informative studies underscore the translational value of this functional knowledge. Structure-derived annotations identify hundreds of high-confidence candidates for mechanisms of antibiotic resistance, virulence factors, and basic metabolism and other functions key in clinical and basic tuberculosis research. More broadly, they provide a systematic framework for improving prokaryotic reference annotations.

Pyrazinamide Susceptibility Is Driven by Activation of the SigE-Dependent Cell Envelope Stress Response in Mycobacterium tuberculosis.

Pyrazinamide (PZA) plays a crucial role in first-line tuberculosis drug therapy. Unlike other antimicrobial agents, PZA is active against Mycobacterium tuberculosis only at low pH. The basis for this conditional drug susceptibility remains undefined. In this study, we utilized a genome-wide approach to interrogate potentiation of PZA action. We found that mutations in numerous genes involved in central metabolism as well as cell envelope maintenance and stress response are associated with PZA resistance. Further, we demonstrate that constitutive activation of the cell envelope stress response can drive PZA susceptibility independent of environmental pH. Consequently, exposure to peptidoglycan synthesis inhibitors, such as beta-lactams and d-cycloserine, potentiate PZA action through triggering this response. These findings illuminate a regulatory mechanism for conditional PZA susceptibility and reveal new avenues for enhancing potency of this important drug through targeting activation of the cell envelope stress response. IMPORTANCE For decades, pyrazinamide has served as a cornerstone of tuberculosis therapy. Unlike any other antitubercular drug, pyrazinamide requires an acidic environment to exert its action. Despite its importance, the driver of this conditional susceptibility has remained unknown. In this study, a genome-wide approach revealed that pyrazinamide action is governed by the cell envelope stress response. This observation was validated by orthologous approaches that demonstrate that a central player of this response, SigE, is both necessary and sufficient for potentiation of pyrazinamide action. Moreover, constitutive activation of this response through deletion of the anti-sigma factor gene rseA or exposure of bacilli to drugs that target the cell wall was found to potently drive pyrazinamide susceptibility independent of environmental pH. These findings force a paradigm shift in our understanding of pyrazinamide action and open new avenues for improving diagnostic and therapeutic tools for tuberculosis.

Genetic Determinants Enabling Medium-Dependent Adaptation to Nafcillin in Methicillin-Resistant Staphylococcus aureus.

Antimicrobial susceptibility testing standards driving clinical decision-making have centered around the use of cation-adjusted Mueller-Hinton broth (CA-MHB) as the medium with the notion of supporting bacterial growth, without consideration of recapitulating the in vivo environment. However, it is increasingly recognized that various medium conditions have tremendous influence on antimicrobial activity, which in turn may have major implications on the ability of in vitro susceptibility assays to predict antibiotic activity in vivo. To elucidate differential growth optimization and antibiotic resistance mechanisms, adaptive laboratory evolution was performed in the presence or absence of the antibiotic nafcillin with methicillin-resistant Staphylococcus aureus (MRSA) TCH1516 in either (i) CA-MHB, a traditional bacteriological nutritionally rich medium, or (ii) Roswell Park Memorial Institute (RPMI), a medium more reflective of the in vivo host environment. Medium adaptation analysis showed an increase in growth rate in RPMI, but not CA-MHB, with mutations in apt, adenine phosphoribosyltransferase, and the manganese transporter subunit, mntA, occurring reproducibly in parallel replicate evolutions. The medium-adapted strains showed no virulence attenuation. Continuous exposure of medium-adapted strains to increasing concentrations of nafcillin led to medium-specific evolutionary strategies. Key reproducibly occurring mutations were specific for nafcillin adaptation in each medium type and did not confer resistance in the other medium environment. Only the vraRST operon, a regulator of membrane- and cell wall-related genes, showed mutations in both CA-MHB- and RPMI-evolved strains. Collectively, these results demonstrate the medium-specific genetic adaptive responses of MRSA and establish adaptive laboratory evolution as a platform to study clinically relevant resistance mechanisms.IMPORTANCE The ability of pathogens such as Staphylococcus aureus to evolve resistance to antibiotics used in the treatment of infections has been an important concern in the last decades. Resistant acquisition usually translates into treatment failure and puts patients at risk of unfavorable outcomes. Furthermore, the laboratory testing of antibiotic resistance does not account for the different environment the bacteria experiences within the human body, leading to results that do not translate into the clinic. In this study, we forced methicillin-resistant S. aureus to develop nafcillin resistance in two different environments, a laboratory environment and a physiologically more relevant environment. This allowed us to identify genetic changes that led to nafcillin resistance under both conditions. We concluded that not only does the environment dictate the evolutionary strategy of S. aureus to nafcillin but also that the evolutionary strategy is specific to that given environment.

The Bewildering Antitubercular Action of Pyrazinamide.

Pyrazinamide (PZA) is a cornerstone antimicrobial drug used exclusively for the treatment of tuberculosis (TB). Due to its ability to shorten drug therapy by 3 months and reduce disease relapse rates, PZA is considered an irreplaceable component of standard first-line short-course therapy for drug-susceptible TB and second-line treatment regimens for multidrug-resistant TB. Despite over 60 years of research on PZA and its crucial role in current and future TB treatment regimens, the mode of action of this unique drug remains unclear. Defining the mode of action for PZA will open new avenues for rational design of novel therapeutic approaches for the treatment of TB. In this review, we discuss the four prevailing models for PZA action, recent developments in modulation of PZA susceptibility and resistance, and outlooks for future research and drug development.

Anti-tubercular Activity of Pyrazinamide is Independent of trans-Translation and RpsA.

Pyrazinamide (PZA) is a first line anti-tubercular drug for which the mechanism of action remains unresolved. Recently, it was proposed that the active form of PZA, pyrazinoic acid (POA), disrupts the ribosome rescue process of trans-translation in Mycobacterium tuberculosis. This model suggested that POA binds within the carboxy-terminal domain of ribosomal protein S1 (RpsA) and inhibits trans-translation leading to accumulation of stalled ribosomes. Here, we demonstrate that M. tuberculosis RpsA interacts with single stranded RNA, but not with POA. Further, we show that an rpsA polymorphism previously identified in a PZA resistant strain does not confer PZA resistance when reconstructed in a laboratory strain. Finally, by utilizing an in vitro trans-translation assay with purified M. tuberculosis ribosomes we find that an interfering oligonucleotide can inhibit trans-translation, yet POA does not inhibit trans-translation. Based on these findings, we conclude that the action of PZA is entirely independent of RpsA and trans-translation in M. tuberculosis.

Long-Chain Fatty Acyl Coenzyme A Ligase FadD2 Mediates Intrinsic Pyrazinamide Resistance in Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a first-line tuberculosis (TB) drug that has been in clinical use for 60 years yet still has an unresolved mechanism of action. Based upon the observation that the minimum concentration of PZA required to inhibit the growth of Mycobacterium tuberculosis is approximately 1,000-fold higher than that of other first-line drugs, we hypothesized that M. tuberculosis expresses factors that mediate intrinsic resistance to PZA. To identify genes associated with intrinsic PZA resistance, a library of transposon-mutagenized Mycobacterium bovis BCG strains was screened for strains showing hypersusceptibility to the active form of PZA, pyrazinoic acid (POA). Disruption of the long-chain fatty acyl coenzyme A (CoA) ligase FadD2 enhanced POA susceptibility by 16-fold on agar medium, and the wild-type level of susceptibility was restored upon expression of fadD2 from an integrating mycobacterial vector. Consistent with the recent observation that POA perturbs mycobacterial CoA metabolism, the fadD2 mutant strain was more vulnerable to POA-mediated CoA depletion than the wild-type strain. Ectopic expression of the M. tuberculosis pyrazinamidase PncA, necessary for conversion of PZA to POA, in the fadD2 transposon insertion mutant conferred at least a 16-fold increase in PZA susceptibility under active growth conditions in liquid culture at neutral pH. Importantly, deletion of fadD2 in M. tuberculosis strain H37Rv also resulted in enhanced susceptibility to POA. These results indicate that FadD2 is associated with intrinsic PZA and POA resistance and provide a proof of concept for the target-based potentiation of PZA activity in M. tuberculosis.

Uncoupling Environmental pH and Intrabacterial Acidification from Pyrazinamide Susceptibility in Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a first-line antitubercular drug for which the mode of action remains unresolved. Mycobacterium tuberculosis lacks measurable susceptibility to PZA under standard laboratory growth conditions. However, susceptibility to this drug can be induced by cultivation of the bacilli in an acidified growth medium. Previous reports suggested that the active form of PZA, pyrazinoic acid (POA), operates as a proton ionophore that confers cytoplasmic acidification when M. tuberculosis is exposed to an acidic environment. In this study, we demonstrate that overexpression of the PZA-activating enzyme PncA can confer PZA susceptibility to M. tuberculosis under neutral and even alkaline growth conditions. Furthermore, we find that wild-type M. tuberculosis displays increased susceptibility to POA relative to PZA in neutral and alkaline media. Utilizing a strain of M. tuberculosis that expresses a pH-sensitive green fluorescent protein (GFP), we find that unlike the bona fide ionophores monensin and carbonyl cyanide 3-chlorophenylhydrazone, PZA and POA do not induce rapid uncoupling or cytoplasmic acidification under conditions that promote susceptibility. Thus, based on these observations, we conclude that the antitubercular action of POA is independent of environmental pH and intrabacterial acidification.

Pantothenate and pantetheine antagonize the antitubercular activity of pyrazinamide.

Pyrazinamide (PZA) is a first-line tuberculosis drug that inhibits the growth of Mycobacterium tuberculosis via an as yet undefined mechanism. An M. tuberculosis laboratory strain that was auxotrophic for pantothenate was found to be insensitive to PZA and to the active form, pyrazinoic acid (POA). To determine whether this phenotype was strain or condition specific, the effect of pantothenate supplementation on PZA activity was assessed using prototrophic strains of M. tuberculosis. It was found that pantothenate and other ß-alanine-containing metabolites abolished PZA and POA susceptibility, suggesting that POA might selectively target pantothenate synthesis. However, when the pantothenate-auxotrophic strain was cultivated using a subantagonistic concentration of pantetheine in lieu of pantothenate, susceptibility to PZA and POA was restored. In addition, we found that ß-alanine could not antagonize PZA and POA activity against the pantothenate-auxotrophic strain, indicating that the antagonism is specific to pantothenate. Moreover, pantothenate-mediated antagonism was observed for structurally related compounds, including n-propyl pyrazinoate, 5-chloropyrazinamide, and nicotinamide, but not for nicotinic acid or isoniazid. Taken together, these data demonstrate that while pantothenate can interfere with the action of PZA, pantothenate synthesis is not directly targeted by PZA. Our findings suggest that targeting of pantothenate synthesis has the potential to enhance PZA efficacy and possibly to restore PZA susceptibility in isolates with panD-linked resistance.

Small-scale, semi-automated purification of eukaryotic proteins for structure determination.

A simple approach that allows cost-effective automated purification of recombinant proteins in levels sufficient for functional characterization or structural studies is described. Studies with four human stem cell proteins, an engineered version of green fluorescent protein, and other proteins are included. The method combines an expression vector (pVP62K) that provides in vivo cleavage of an initial fusion protein, a factorial designed auto-induction medium that improves the performance of small-scale production, and rapid, automated metal affinity purification of His8-tagged proteins. For initial small-scale production screening, single colony transformants were grown overnight in 0.4 ml of auto-induction medium, produced proteins were purified using the Promega Maxwell 16, and purification results were analyzed by Caliper LC90 capillary electrophoresis. The yield of purified [U-15N]-His8-Tcl-1 was 7.5 microg/ml of culture medium, of purified [U-15N]-His8-GFP was 68 microg/ml, and of purified selenomethione-labeled AIA-GFP (His8 removed by treatment with TEV protease) was 172 microg/ml. The yield information obtained from a successful automated purification from 0.4 ml was used to inform the decision to scale-up for a second meso-scale (10-50 ml) cell growth and automated purification. 1H-15N NMR HSQC spectra of His8-Tcl-1 and of His8-GFP prepared from 50 ml cultures showed excellent chemical shift dispersion, consistent with well folded states in solution suitable for structure determination. Moreover, AIA-GFP obtained by proteolytic removal of the His8 tag was subjected to crystallization screening, and yielded crystals under several conditions. Single crystals were subsequently produced and optimized by the hanging drop method. The structure was solved by molecular replacement at a resolution of 1.7 A. This approach provides an efficient way to carry out several key target screening steps that are essential for successful operation of proteomics pipelines with eukaryotic proteins: examination of total expression, determination of proteolysis of fusion tags, quantification of the yield of purified protein, and suitability for structure determination.