Mehmet A. Orman

Metabolic Control of Persister Formation in Escherichia coli

Bacterial persisters are phenotypic variants that form from the action of stress response pathways triggering toxin-mediated antibiotic tolerance. Although persisters form during normal growth from native stresses, the pathways responsible for this phenomenon remain elusive. Here we have discovered that carbon source transitions stimulate the formation of fluoroquinolone persisters in Escherichia coli. Further, through a combination of genetic, biochemical, and flow cytometric assays in conjunction with a mathematical model, we have reconstructed a molecular-level persister formation pathway from initial stress (glucose exhaustion) to the activation of a metabolic toxin-antitoxin (TA) module (the ppGpp biochemical network) resulting in inhibition of DNA gyrase activity, the primary target of fluoroquinolones. This pathway spans from initial stress to antibiotic target and demonstrates that TA behavior can be exhibited by a metabolite-enzyme interaction (ppGpp-SpoT), in contrast to classical TA systems that involve only protein and/or RNA.

Dormancy Is Not Necessary or Sufficient for Bacterial Persistence

ABSTRACT The antibiotic tolerances of bacterial persisters have been attributed to transient dormancy. While persisters have been observed to be growth inhibited prior to antibiotic exposure, we sought to determine whether such a trait was essential to the phenotype. Furthermore, we sought to provide direct experimental evidence of the persister metabolic state so as to determine whether the common assumption of metabolic inactivity was valid. Using fluorescence-activated cell sorting (FACS), a fluorescent indicator of cell division, a fluorescent measure of metabolic activity, and persistence assays, we found that bacteria that are rapidly growing prior to antibiotic exposure can give rise to persisters and that a lack of replication or low metabolic activity prior to antibiotic treatment simply increases the likelihood that a cell is a persister. Interestingly, a lack of significant growth or metabolic activity does not guarantee persistence, as the majority of even “dormant” subpopulations (>99%) were not persisters. These data suggest that persistence is far more complex than dormancy and point to additional characteristics needed to define the persister phenotype.

The role of metabolism in bacterial persistence

Bacterial persisters are phenotypic variants with extraordinary tolerances toward antibiotics. Persister survival has been attributed to inhibition of essential cell functions during antibiotic stress, followed by reversal of the process and resumption of growth upon removal of the antibiotic. Metabolism plays a critical role in this process, since it participates in the entry, maintenance, and exit from the persister phenotype. Here, we review the experimental evidence that demonstrates the importance of metabolism to persistence, highlight the successes and potential of targeting metabolism in the search for anti-persister therapies, and discuss the current methods and challenges to understand persister physiology.

Establishment of a Method To Rapidly Assay Bacterial Persister Metabolism

ABSTRACT Bacterial persisters exhibit an extraordinary tolerance to antibiotics that is dependent on their metabolic state. Although persister metabolism promises to be a rich source of antipersister strategies, there is relatively little known about the metabolism of these rare and transient phenotypic variants. To address this knowledge gap, we explored the use of several techniques, and we found that only one measured persister metabolism. This assay was based on the phenomenon of metabolite-enabled aminoglycoside killing of persisters, and we used it to characterize the metabolic heterogeneity of different persister populations. From these investigations, we determined that glycerol and glucose are the most ubiquitously used carbon sources by various types of Escherichia coli persisters, suggesting that these metabolites might prove beneficial to deliver in conjunction with aminoglycosides for the treatment of chronic and recurrent infections. In addition, we demonstrated that the persister metabolic assay developed here is amenable to high-throughput screening with the use of phenotype arrays.

Inhibition of stationary phase respiration impairs persister formation in E. coli.

Bacterial persisters are rare phenotypic variants that temporarily tolerate high antibiotic concentrations. Persisters have been hypothesized to underlie the recalcitrance of biofilm infections, and strategies to eliminate these cells have the potential to improve treatment outcomes for many hospital-treated infections. Here we investigate the role of stationary phase metabolism in generation of type I persisters in Escherichia coli, which are those that are formed by passage through stationary phase. We find that persisters are unlikely to derive from bacteria with low redox activity, and that inhibition of respiration during stationary phase reduces persister levels by up to ∼1,000-fold. Loss of stationary phase respiratory activity prevents digestion of endogenous proteins and RNA, which yields bacteria that are more capable of translation, replication and concomitantly cell death when exposed to antibiotics. These findings establish bacterial respiration as a prime target for reducing the number of persisters formed in nutrient-depleted, non-growing populations.

The influence of carbon sources on recombinant‐human‐ growth‐hormone production by Pichia pastoris is dependent on phenotype: a comparison of Muts and Mut+ strains

The influence of carbon sources on rhGH (recombinant human growth hormone) production by two Pichia pastoris strains having different methanol utilization phenotypes (P. pastoris‐hGH‐Mut+ and P. pastoris‐hGH‐Muts) was investigated using batch bioreactors. The effect of methanol concentration (CMeOH) in defined and complex media, and further glycerol/methanol mixed defined media, was analysed systematically over a wide range. With methanol as the sole carbon source, strain Muts grew only slightly, whereas with Mut+, a cell concentration (CX) of 6.0 g of dry cells/dm3 was obtained and an rhGH concentration (CrhGH) of 0.032 g/dm3 was produced. In complex medium without glycerol at a CMeOH of 2% (v/v), a CrhGH of 0.16 g of rhGH/dm3 was produced by Muts, a value 3‐fold higher than that produced by Mut+, despite the fact that the CX of Mut+ (6.1 g/dm3) was 2‐fold higher than that of Muts (3.0 g/dm3). In a glycerol/methanol mixed defined medium, methanol consumption began when glycerol was totally depleted, indicating that glycerol is a repressor of the AOX1 (alcohol oxidase‐1 gene) promoter. With strain Muts at a glycerol concentration (CGly) of 30 g/dm3 and a CMeOH of 1% (v/v), the CrhGH produced was 0.11 g/dm3, whereas, with the Mut+ strain, a CrhGH of 0.06 g/dm3 was obtained at a CGly of 30 g/dm3 and a CMeOH of 4%. As methanol is not consumed by Muts strain effectively and the presence of methanol in the fermentation broth triggers induction of the AOX1 promoter, our results encourage the use of the Muts strain for rhGH production. In addition to rhGH production, the specific cell growth rates, specific methanol and/or glycerol utilization rates and maintenance coefficients in methanol‐ and glycerol‐based defined media were determined. With a methanol‐based defined medium and using the Mut+ strain, a higher specific growth rate (μ) of approx. 0.14 h−1 was observed during the exponential cell growth phase at a CMeOH of ≤2.0%. When glycerol was used as a sole carbon source, both phenotypes showed similar cell‐growth and glycerol‐utilization rates. The results of the present study should enable one to optimize the expression of other therapeutic proteins by P. pastoris.

Expression System for Synthesis and Purification of Recombinant Human Growth Hormone in Pichia pastoris and Structural Analysis by MALDI-ToF Mass Spectrometry

An expression system in Pichia pastoris for the production and purification of recombinant human growth hormone (rHGH) was designed and implemented. hGH cDNA sequence was cloned into pPICZαA vector under the control of AOX1 promoter, which included a polyhistidine‐tag on the amino terminal end to enable affinity purification and a target site for Factor Xa protease such that protease cleavage in vitro would produce rhGH without any non‐native N‐and C‐termini. Analyses of the affinity‐purified rhGH product by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) showed a spectral peak at m/ z 23699. Purified product digested with Factor Xa protease had a molecular mass of 22132 kDa. The molecular mass difference before and after Factor Xa protease digestion expectedly corresponds to the 12 amino acids in the rhGH amino terminus, which includes the EcoRI digestion site (Glu‐Phe), the 6xHis tag for affinity purification, and the Factor Xa protease recognition sequence (Ile‐Glu‐Gly‐Arg), a result that also indicates that the signal peptide was properly processed by P. pastoris. N‐Terminal sequence analysis of the Factor Xa protease trimmed recombinant product confirmed the mature hGH sequence. Thus, the system designed functioned with its intended purpose effectively in expression, cleavage, and purification of the recombinant product.

Pathway analysis of liver metabolism under stressed condition

Pathway analysis is a useful tool which reveals important metabolic network properties. However, the big challenge is to propose an objective function for estimating active pathways, which represent the actual state of network. In order to provide weight values for all possible pathways within the metabolic network, this study presents different approaches, considering the structural and physiological properties of the metabolic network, aiming at a unique decomposition of the flux vector into pathways. These methods were used to analyze the hepatic metabolism considering available data sets obtained from the perfused livers of fasted rats receiving burn injury. Utilizing unique decomposition techniques and different fluxes revealed that higher weights were always attributed to short pathways. Specific pathways, including pyruvate, glutamate and oxaloacetate pools, and urea production from arginine, were found to be important or essential in all methods and experimental conditions. Moreover the pathways, including serine production from glycine and conversion between acetoacetate and B-OH-butyrate, were assigned higher weights. Pathway analysis was also used to identify the main sources for the production of certain products in the hepatic metabolic network to gain a better understanding of the effects of burn injury on liver metabolism.

Metabolic flux determination in perfused livers by mass balance analysis: Effect of fasting

Isolated perfused livers from fasted rats have been extensively studied to understand the underlying mechanisms of injury‐induced acute and chronic changes in liver metabolism. In this study, we investigated the impact of fasting on perfused liver metabolism. The methodology we used combines a flux variability approach, sampling analysis and singular value decomposition (SVD) to analyze the data using the same metabolic reaction network for both fed and fasted livers. Considering both experimental observations and mathematical analysis, the results show that 24 h fasting results in a limited glucose production from glycogen and up‐regulation of gluconeogenic pathway. Glutamate metabolism and fatty acid oxidation are also slightly up‐regulated whereas aspartate metabolism is down‐regulated after fasting. Moreover, SVD analysis elucidates that glycogen breakdown mainly affects the pentose phosphate pathway in fasted state whereas co‐occurrence pattern associated with glycogen breakdown and glycolysis observed in flux samples of fed state is dominant. In conclusion, this analysis provides a detailed description of the metabolic changes in two different states, considering the entire steady state flux space and dominant flux patterns that capture the system variations within the flux space. Biotechnol. Bioeng. 2010;107: 825–835.

The dynamics of the early inflammatory response in double-hit burn and sepsis animal models

Severe burn trauma is generally associated with bacterial infections, which causes a more persistent inflammatory response with an ongoing hypermetabolic and catabolic state. This complex biological response, mediated by chemokines and cytokines, can be more severe when excessive interactions between the mediators take place. In this study, the early inflammatory response following the cecum ligation and puncture (CLP) or its corresponding control treatment (sham-CLP or SCLP) in burn (B) male rats was analyzed by measuring 23 different cytokines and chemokines. Cytokines and chemokines, including MCP-1, IP-10, leptin, TNF-α, MIP-1α, IL-18, GMCSF, RANTES and GCSF were significantly altered in both B+CLP and B+SCLP groups. IL-10 and IL-6 were significantly up-regulated in the B+CLP group when compared to the B+SCLP group. Down regulation of leptin and IP-10 concentrations were found to be related to surgery and/or infection. IL-18 and MCP-1 were elevated in all groups including previously published single injury models receiving similar treatments. In this study, insult-specific mediators with their characteristic temporal patterns were elucidated in double hit models.