An integrated network analysis reveals that nitric oxide reductase prevents metabolic cycling of nitric oxide by Pseudomonas aeruginosa.

Nitric oxide (NO) is a chemical weapon within the arsenal of immune cells, but is also generated endogenously by different bacteria. Pseudomonas aeruginosa are pathogens that contain an NO-generating nitrite (NO2-) reductase (NirS), and NO has been shown to influence their virulence. Interestingly, P. aeruginosa also contain NO dioxygenase (Fhp) and nitrate (NO3-) reductases, which together with NirS provide the potential for NO to be metabolically cycled (NO→NO3-→NO2-→NO). Deeper understanding of NO metabolism in P. aeruginosa will increase knowledge of its pathogenesis, and computational models have proven to be useful tools for the quantitative dissection of NO biochemical networks. Here we developed such a model for P. aeruginosa and confirmed its predictive accuracy with measurements of NO, O2, NO2-, and NO3- in mutant cultures devoid of Fhp or NorCB (NO reductase) activity. Using the model, we assessed whether NO was metabolically cycled in aerobic P. aeruginosa cultures. Calculated fluxes indicated a bottleneck at NO3-, which was relieved upon O2 depletion. As cell growth depleted dissolved O2 levels, NO3- was converted to NO2- at near-stoichiometric levels, whereas NO2- consumption did not coincide with NO or NO3- accumulation. Assimilatory NO2- reductase (NirBD) or NorCB activity could have prevented NO cycling, and experiments with ΔnirB, ΔnirS, and ΔnorC showed that NorCB was responsible for loss of flux from the cycle. Collectively, this work provides a computational tool to analyze NO metabolism in P. aeruginosa, and establishes that P. aeruginosa use NorCB to prevent metabolic cycling of NO.

Ploidy is an important determinant of fluoroquinolone persister survival.

Genetic mutants have demonstrated the importance of homologous recombination (HR) to fluoroquinolone (FQ) persistence, which suggests that single-cell chromosome (Chr) abundance might be a phenotypic variable of importance to persisters. Here, we sorted stationary-phase E. coli based on ploidy and subjected the subpopulations to tolerance assays. Subpopulations sorted to contain diploid cells harbored up to ∼40-fold more FQ persisters than those sorted to contain monoploid cells. This association was observed with distinct FQs, in independent environmental conditions, and with more than one strain of E. coli (MG1655; uropathogenic CFT073) but was abolished in HR-deficient strains (ΔrecA and ΔrecB). It was observed that the persister level of monoploid subpopulations exceeded those of ΔrecA and ΔrecB by 10-fold or more, and subsequent high-purity sorting confirmed that observation. Those data suggested the existence of distinct FQ persister subtypes: those that are and are not proficient with HR. Time-lapse microscopy revealed significant differences in initial size and growth dynamics during the post-antibiotic recovery period for persisters from monoploid- and diploid-enriched subpopulations. In addition, non-persisters in monoploid-enriched subpopulations elongated minimally following FQ treatment, resembling previous observations of HR-deficient strains, whereas non-persisters in diploid-enriched subpopulations on average filamented extensively. Together, these results identify a phenotypic variable with a significant impact on FQ persistence, establish the existence of more than one type of persister to the same antibiotic in an isogenic culture, and reveal roles for RecA and RecB in FQ persistence, even in the absence of homologous chromosomes.

Counting Chromosomes in Individual Bacteria to Quantify Their Impacts on Persistence.

Persisters are phenotypic variants within bacterial populations that tolerate antibiotic treatments considerably better than the majority of cells. A phenotypic quality that varies within bacterial populations is the chromosome number of individual cells. One, two, four, or more chromosomes per cell have been observed previously, and the impact of genome copy number can range from gene dosage effects to an inability to perform specific DNA repair functions, such as homologous recombination. We hypothesize that chromosome abundance is an underappreciated phenotypic variable that could impact persistence to antibiotics. Here, we describe methodologies to segregate bacterial populations based on chromosome number, assess the purity of those subpopulations, and suggest assays that could be used to quantify the impacts of genome abundance on persistence.

Enhanced antibiotic resistance development from fluoroquinolone persisters after a single exposure to antibiotic.

Bacterial persisters are able to tolerate high levels of antibiotics and give rise to new populations. Persister tolerance is generally attributed to minimally active cellular processes that prevent antibiotic-induced damage, which has led to the supposition that persister offspring give rise to antibiotic-resistant mutants at comparable rates to normal cells. Using time-lapse microscopy to monitor Escherichia coli populations following ofloxacin treatment, we find that persisters filament extensively and induce impressive SOS responses before returning to a normal appearance. Further, populations derived from fluoroquinolone persisters contain significantly greater quantities of antibiotic-resistant mutants than those from untreated controls. We confirm that resistance is heritable and that the enhancement requires RecA, SOS induction, an opportunity to recover from treatment, and the involvement of error-prone DNA polymerase V (UmuDC). These findings show that fluoroquinolones damage DNA in persisters and that the ensuing SOS response accelerates the development of antibiotic resistance from these survivors.

SELEX and SHAPE reveal that sequence motifs and an extended hairpin in the 5' portion of Turnip crinkle virus satellite RNA C mediate fitness in plants.

Noncoding RNAs use their sequence and/or structure to mediate function(s). The 5' portion (166 nt) of the 356-nt noncoding satellite RNA C (satC) of Turnip crinkle virus (TCV) was previously modeled to contain a central region with two stem-loops (H6 and H7) and a large connecting hairpin (H2). We now report that in vivo functional selection (SELEX) experiments assessing sequence/structure requirements in H2, H6, and H7 reveal that H6 loop sequence motifs were recovered at nonrandom rates and only some residues are proposed to base-pair with accessible complementary sequences within the 5' central region. In vitro SHAPE of SELEX winners indicates that the central region is heavily base-paired, such that along with the lower stem and H2 region, one extensive hairpin exists composing the entire 5' region. As these SELEX winners are highly fit, these characteristics facilitate satRNA amplification in association with TCV in plants.

A Systematic Approach for Developing Bacteria-Specific Imaging Tracers.

The modern patient is increasingly susceptible to bacterial infections including those due to multidrug-resistant organisms (MDROs). Noninvasive whole-body analysis with pathogen-specific imaging technologies can significantly improve patient outcomes by rapidly identifying a source of infection and monitoring the response to treatment, but no such technology exists clinically. METHODS: We systematically screened 961 random radiolabeled molecules in silico as substrates for essential metabolic pathways in bacteria, followed by in vitro uptake in representative bacteria-Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and mycobacteria. Fluorine-labeled analogs, that could be developed as PET-based imaging tracers, were evaluated in a murine myositis model. RESULTS: We identified 3 novel, nontoxic molecules demonstrating selective bacterial uptake: para-aminobenzoic acid (PABA), with uptake in all representative bacteria including Mycobacterium tuberculosis; mannitol, with selective uptake in S. aureus and E. coli; and sorbitol, accumulating only in E. coli None accumulated in mammalian cells or heat-killed bacteria, suggesting metabolism-derived specificity. In addition to an extended bacterial panel of laboratory strains, all 3 molecules rapidly accumulated in respective clinical isolates of interest including MDROs such as methicillin-resistant S. aureus, extended-spectrum ß-lactamase-producing, and carbapenem-resistant Enterobacteriaceae. In a murine myositis model, fluorine-labeled analogs of all 3 molecules could rapidly detect and differentiate infection sites from sterile inflammation in mice (P = 0.03). Finally, 2-deoxy-2-[F-18]fluoro-d-sorbitol (18F-FDS) can be easily synthesized from 18F-FDG. PET, with 18F-FDS synthesized using current good manufacturing practice, could rapidly differentiate true infection from sterile inflammation to selectively localize E. coli infection in mice. CONCLUSION: We have developed a systematic approach that exploits unique biochemical pathways in bacteria to develop novel pathogen-specific imaging tracers. These tracers have significant potential for clinical translation to specifically detect and localize a broad range of bacteria, including MDROs.

Rapid evolution of in vivo-selected sequences and structures replacing 20% of a subviral RNA.

The 356 nt noncoding satellite RNA C (satC) of Turnip crinkle virus (TCV) is composed of 5' sequences from a second TCV satRNA (satD) and 3' sequences derived from TCV. SHAPE structure mapping revealed that 76 nt in the poorly-characterized satD-derived region form an extended hairpin (H2). Pools of satC in which H2 was replaced with 76, 38, or 19 random nt were co-inoculated with TCV helper virus onto plants and satC fitness assessed using in vivo functional selection (SELEX). The most functional progeny satCs, including one as fit as wild-type, contained a 38-39 nt H2 region that adopted a hairpin structure and exhibited an increased ratio of dimeric to monomeric molecules. Some progeny of satC with H2 deleted featured a duplication of 38 nt, partially rebuilding the deletion. Therefore, H2 can be replaced by a 38-39 nt hairpin, sufficient for overall structural stability of the 5' end of satC.

Imaging the evolution of reactivation pulmonary tuberculosis in mice using 18F-FDG PET.

UNLABELLED: Latent tuberculosis infection affects one third of the world's population and can reactivate (relapse) decades later. However, current technologies, dependent on postmortem analyses, cannot follow the temporal evolution of disease. METHODS: C3HeB/FeJ mice, which develop necrotic and hypoxic tuberculosis lesions, were aerosol-infected with Mycobacterium tuberculosis. PET and CT were used to serially image the same cohort of infected mice through pretreatment, tuberculosis treatment, and subsequent development of relapse. RESULTS: A novel diffeomorphic registration was successfully used to monitor the spatial evolution of individual pulmonary lesions. Although most lesions during relapse developed in the same regions as those noted during pretreatment, several lesions also arose de novo within regions with no prior lesions. CONCLUSION: This study presents a novel model that simulates infection and reactivation disease as seen in humans and could prove valuable to study tuberculosis pathogenesis and evaluate novel therapeutics.

Imaging Enterobacteriaceae infection in vivo with 18F-fluorodeoxysorbitol positron emission tomography.

The Enterobacteriaceae are a family of rod-shaped Gram-negative bacteria that normally inhabit the gastrointestinal tract and are the most common cause of Gram-negative bacterial infections in humans. In addition to causing serious multidrug-resistant, hospital-acquired infections, a number of Enterobacteriaceae species are also recognized as biothreat pathogens. As a consequence, new tools are urgently needed to specifically identify and localize infections due to Enterobacteriaceae and to monitor antimicrobial efficacy. In this report, we used commercially available 2-[(18)F]-fluorodeoxyglucose ((18)F-FDG) to produce 2-[(18)F]-fluorodeoxysorbitol ((18)F-FDS), a radioactive probe for Enterobacteriaceae, in 30 min. (18)F-FDS selectively accumulated in Enterobacteriaceae, but not in Gram-positive bacteria or healthy mammalian or cancer cells in vitro. In a murine myositis model, (18)F-FDS positron emission tomography (PET) rapidly differentiated true infection from sterile inflammation with a limit of detection of 6.2 ± 0.2 log10 colony-forming units (CFU) for Escherichia coli. Our findings were extended to models of mixed Gram-positive and Gram-negative thigh co-infections, brain infection, Klebsiella pneumonia, and mice undergoing immunosuppressive chemotherapy. This technique rapidly and specifically localized infections due to Enterobacteriaceae, providing a three-dimensional holistic view within the animal. Last, (18)F-FDS PET monitored the efficacy of antimicrobial treatment, demonstrating a PET signal proportionate to the bacterial burden. Therapeutic failures associated with multidrug-resistant, extended-spectrum ß-lactamase (ESBL)-producing E. coli infections were detected in real time. Together, these data show that (18)F-FDS is a candidate imaging probe for translation to human clinical cases of known or suspected infections owing to Enterobacteriaceae.