Repurposing of Anticancer Drugs for the Treatment of Bacterial Infections.

Despite the fact that bacterial infections are one of the leading causes of death worldwide and that mortality rates are increasing at alarming rates, no new antibiotics have been produced by the pharmaceutical industry in more than a decade. The situation is so dire that the World Health Organization warned that we may enter a "post-antibiotic era" within this century; accordingly, bacteria resistant against all known antibiotics are becoming common and already producing untreatable infections. Although several novel approaches to combat bacterial infections have been proposed, they have yet to be implemented in clinical practice. Hence, we propose that a more plausible and faster approach is the utilization of drugs originally developed for other purposes besides antimicrobial activity. Among these are some anticancer molecules proven effective in vitro for eliminating recalcitrant, multidrug tolerant bacteria; some of which also protect animals from infections and recently are undergoing clinical trials. In this review, we highlight the similarities between cancer cells/tumors and bacterial infections, and present evidence that supports the utilization of some anticancer drugs, including 5-fluorouracil (5-FU), gallium (Ga) compounds, and mitomycin C, as antibacterials. Each of these drugs has some promising properties such as broad activity (all three compounds), dual antibiotic and antivirulence properties (5-FU), efficacy against multidrug resistant strains (Ga), and the ability to kill metabolically dormant persister cells which cause chronic infections (mitomycin C).

Antibiotic-tolerant Staphylococcus aureus Biofilm Persists on Arthroplasty Materials.

BACKGROUND: The continued presence of biofilm may be one cause of the high risk of failure observed with irrigation and débridement with component retention in acute periprosthetic joint infection (PJI). There is a poor understanding of the role of biofilm antibiotic tolerance in PJI. QUESTIONS/PURPOSES: (1) Do increasing doses of cefazolin result in decreased viable biofilm mass on arthroplasty materials? (2) Is cefazolin resistance phenotypic or genotypic? (3) Is biofilm viability a function of biofilm depth after treatment with cefazolin? (4) Is the toxin-antitoxin system, yoeB expression, associated with antibiotic stress? METHODS: Methicillin-sensitive Staphylococcus aureus biofilm was cultured on total knee arthroplasty (TKA) materials and exposed to increasing doses of cefazolin (control, 0.5, 1.0, 10.0, 100.0 µg/mL). Quantitative confocal microscopy and quantitative culture were used to measure viable biofilm cell density. To determine if cefazolin resistance was phenotypic or genotypic, we measured minimum inhibitory concentration (MIC) after exposure to different cefazolin concentrations; changes in MIC would suggest genotypic features, whereas unchanged MIC would suggest phenotypic behavior. Finally, quantitative reverse transcription-polymerase chain reaction was used to quantify expression of yoeB levels between biofilm and planktonic bacteria after exposure to 1 µg/mL cefazolin for 3 hours. RESULTS: Although live biofilm mass was reduced by exposure to cefazolin when compared with biofilm mass in controls (39.2 × 10(3) ± 26.4 × 10(3) pixels), where the level after 0.5 µg/mL exposure also showed reduced mass (20.3 × 10(3) ± 11.9 × 10(3) pixels), no further reduction was seen after higher doses (mass at 1.0 µg/mL: 5.0 × 10(3) pixels ± 1.1 × 10(3) pixels; at 10.0 µg/mL: 6.4 × 10(3) ± 9.6 × 10(3) pixels; at 100.0 µg/mL: 6.4 × 10(3) ± 3.9 × 10(3)). At the highest concentration tested (100 µg/mL), residual viable biofilm was present on all three materials, and there were no differences in percent biofilm survival among cobalt-chromium (18.5% ± 15.1%), polymethylmethacrylate (22.8% ± 20.2%), and polyethylene (14.7% ± 10.4%). We found that tolerance was a phenotypic phenomenon, because increasing cefazolin exposure did not result in changes in MIC as compared with controls (MIC in controls: 0.13 ± 0.02; at 0.5 µg/mL: 0.13 ± 0.001, p = 0.96; at 1.0 µg/m: 0.14 ± 0.04, p = 0.95; at 10.0 µg/m: 0.11 ± 0.016, p = 0.47; at 100.0 µg/m: 0.94 ± 0.047, p = 0.47). Expression of yoeB after 1 µg/mL cefazolin for 3 hours in biofilm cells was greater in biofilm but not in planktonic cells (biofilm: 62.3-fold change, planktonic cells: -78.8-fold change, p < 0.001). CONCLUSIONS: Antibiotics are inadequate at complete removal of the biofilm from the surface of TKA materials. Results suggest that bacterial persisters are responsible for this phenotypic behavior allowing biofilm high tolerance to antibiotics. CLINICAL RELEVANCE: Antibiotic-tolerant biofilm suggests a mechanism behind the poor results in irrigation and débridement for acute TKA PJI.

Toxin MqsR cleaves single-stranded mRNA with various 5' ends.

Toxin/antitoxin (TA) systems are the means by which bacterial cells become persistent; that is, those cells that are tolerant to multiple environmental stresses such as antibiotics by becoming metabolically dormant. These persister cells are responsible for recalcitrant infections. Once toxins are activated by the inactivation of antitoxins (e.g., stress-triggered Lon degradation of the antitoxin), many toxins reduce metabolism by inhibiting translation (e.g., cleaving mRNA, reducing ATP). The MqsR/MqsA TA system of Escherichia coli cleaves mRNA to help the cell withstand oxidative and bile acid stress. Here, we investigated the role of secondary structure and 5' mRNA processing on MqsR degradation of mRNA and found that MqsR cleaves only single-stranded RNA at 5'-GCU sites and that MqsR is equally active against RNA with 5'-triphosphate, 5'-monophosphate, and 5'-hydroxyl groups.

Persistence Increases in the Absence of the Alarmone Guanosine Tetraphosphate by Reducing Cell Growth.

Most bacterial cells are stressed, and as a result, some become tolerant to antibiotics by entering a dormant state known as persistence. The key intracellular metabolite that has been linked to this persister state is guanosine tetraphosphate (ppGpp), the alarmone that was first linked to nutrient stress. In Escherichia coli, ppGpp redirects protein production during nutrient stress by interacting with RNA polymerase directly and by inhibiting several proteins. Consistently, increased levels of ppGpp lead to increased persistence; but, the mechanism by which elevated ppGpp translates into persistence has not been determined. Hence, we explored persistence in the absence of ppGpp so that the underlying mechanism of persister cell formation could be explored. We found that persister cells still form, although at lower levels, in the absence of ppGpp. Additionally, the toxin/antitoxin systems that we investigated (MqsR, MazF, GhoT, and YafQ) remain able to increase persistence dramatically in the absence of ppGpp. By overproducing each E. coli protein from the 4287 plasmid vectors of the ASKA library and selecting for increased persistence in the absence of ppGpp (via a relA spoT mutant), we identified five new proteins, YihS, PntA, YqjE, FocA, and Zur, that increase persistence simply by reducing cell growth.

Combatting bacterial infections by killing persister cells with mitomycin C.

Persister cells are a multi-drug tolerant subpopulation of bacteria that contribute to chronic and recalcitrant clinical infections such as cystic fibrosis and tuberculosis. Persisters are metabolically dormant, so they are highly tolerant to all traditional antibiotics which are mainly effective against actively growing cells. Here, we show that the FDA-approved anti-cancer drug mitomycin C (MMC) eradicates persister cells through a growth-independent mechanism. MMC is passively transported and bioreductively activated, leading to spontaneous cross-linking of DNA, which we verify in both active and dormant cells. We find MMC effectively eradicates cells grown in numerous different growth states (e.g. planktonic cultures and highly robust biofilm cultures) in both rich and minimal media. Additionally, MMC is a potent bactericide for a broad range of bacterial persisters, including commensal Escherichia coli K-12 as well as pathogenic species of E. coli, Staphylococcus aureus and Pseudomonas aeruginosa. We also demonstrate the efficacy of MMC in an animal model and a wound model, substantiating the clinical applicability of MMC against bacterial infections. Therefore, MMC is the first broad-spectrum compound capable of eliminating persister cells, meriting investigation as a new approach for the treatment of recalcitrant infections.

Toxin YafQ increases persister cell formation by reducing indole signalling.

Persister cells survive antibiotic and other environmental stresses by slowing metabolism. Since toxins of toxin/antitoxin (TA) systems have been postulated to be responsible for persister cell formation, we investigated the influence of toxin YafQ of the YafQ/DinJ Escherichia coli TA system on persister cell formation. Under stress, YafQ alters metabolism by cleaving transcripts with in-frame 5'-AAA-G/A-3' sites. Production of YafQ increased persister cell formation with multiple antibiotics, and by investigating changes in protein expression, we found that YafQ reduced tryptophanase levels (TnaA mRNA has 16 putative YafQ cleavage sites). Consistently, TnaA mRNA levels were also reduced by YafQ. Tryptophanase is activated in the stationary phase by the stationary-phase sigma factor RpoS, which was also reduced dramatically upon production of YafQ. Tryptophanase converts tryptophan into indole, and as expected, indole levels were reduced by the production of YafQ. Corroborating the effect of YafQ on persistence, addition of indole reduced persistence. Furthermore, persistence increased upon deleting tnaA, and persistence decreased upon adding tryptophan to the medium to increase indole levels. Also, YafQ production had a much smaller effect on persistence in a strain unable to produce indole. Therefore, YafQ increases persistence by reducing indole, and TA systems are related to cell signalling.

Phosphodiesterase DosP increases persistence by reducing cAMP which reduces the signal indole.

Persisters are bacteria that are highly tolerant to antibiotics due to their dormant state and are of clinical significance owing to their role in infections. Given that the population of persisters increases in biofilms and that cyclic diguanylate (c-di-GMP) is an intracellular signal that increases biofilm formation, we sought to determine whether c-di-GMP has a role in bacterial persistence. By examining the effect of 30 genes from Escherichia coli, including diguanylate cyclases that synthesize c-di-GMP and phosphodiesterases that breakdown c-di-GMP, we determined that DosP (direct oxygen sensing phosphodiesterase) increases persistence by over a thousand fold. Using both transcriptomic and proteomic approaches, we determined that DosP increases persistence by decreasing tryptophanase activity and thus indole. Corroborating this effect, addition of indole reduced persistence. Despite the role of DosP as a c-di-GMP phosphodiesterase, the decrease in tryptophanase activity was found to be a result of cyclic adenosine monophosphate (cAMP) phosphodiesterase activity. Corroborating this result, the reduction of cAMP via CpdA, a cAMP-specific phosphodiesterase, increased persistence and reduced indole levels similarly to DosP. Therefore, phosphodiesterase DosP increases persistence by reducing the interkingdom signal indole via reduction of the global regulator cAMP.

The MqsR/MqsA toxin/antitoxin system protects Escherichia coli during bile acid stress.

Toxin/antitoxin (TA) systems are ubiquitous within bacterial genomes, and the mechanisms of many TA systems are well characterized. As such, several roles for TA systems have been proposed, such as phage inhibition, gene regulation and persister cell formation. However, the significance of these roles is nebulous due to the subtle influence from individual TA systems. For example, a single TA system has only a minor contribution to persister cell formation. Hence, there is a lack of defining physiological roles for individual TA systems. In this study, phenotype assays were used to determine that the MqsR/MqsA type II TA system of Escherichia coli is important for cell growth and tolerance during stress from the bile salt deoxycholate. Using transcriptomics and purified MqsR, we determined that endoribonuclease toxin MqsR degrades YgiS mRNA, which encodes a periplasmic protein that promotes deoxycholate uptake and reduces tolerance to deoxycholate exposure. The importance of reducing YgiS mRNA by MqsR is evidenced by improved growth, reduced cell death and reduced membrane damage when cells without ygiS are stressed with deoxycholate. Therefore, we propose that MqsR/MqsA is physiologically important for E. coli to thrive in the gallbladder and upper intestinal tract, where high bile concentrations are prominent.

Quorum sensing enhancement of the stress response promotes resistance to quorum quenching and prevents social cheating.

Quorum sensing (QS) coordinates the expression of virulence factors and allows bacteria to counteract the immune response, partly by increasing their tolerance to the oxidative stress generated by immune cells. Despite the recognized role of QS in enhancing the oxidative stress response, the consequences of this relationship for the bacterial ecology remain unexplored. Here we demonstrate that QS increases resistance also to osmotic, thermal and heavy metal stress. Furthermore a QS-deficient lasR rhlR mutant is unable to exert a robust response against H2O2 as it has less induction of catalase and NADPH-producing dehydrogenases. Phenotypic microarrays revealed that the mutant is very sensitive to several toxic compounds. As the anti-oxidative enzymes are private goods not shared by the population, only the individuals that produce them benefit from their action. Based on this premise, we show that in mixed populations of wild-type and the mexR mutant (resistant to the QS inhibitor furanone C-30), treatment with C-30 and H2O2 increases the proportion of mexR mutants; hence, oxidative stress selects resistance to QS compounds. In addition, oxidative stress alone strongly selects for strains with active QS systems that are able to exert a robust anti oxidative response and thereby decreases the proportion of QS cheaters in cultures that are otherwise prone to invasion by cheats. As in natural environments stress is omnipresent, it is likely that this QS enhancement of stress tolerance allows cells to counteract QS inhibition and invasions by social cheaters, therefore having a broad impact in bacterial ecology.

de novo synthesis of a bacterial toxin/antitoxin system.

The prevalence of toxin/antitoxin (TA) systems in almost all genomes suggests they evolve rapidly. Here we show that an antitoxin from a type V system (GhoS, an endoribonuclease specific for the mRNA of the toxin GhoT) can be converted into a novel toxin (ArT) simply by adding two mutations. In contrast to GhoS, which increases growth, the new toxin ArT decreases growth dramatically in Escherichia coli. Transmission electron microscopy analysis revealed that the nucleoid in ArT-producing cells is concentrated and appears hollow. Whole-transcriptome profiling revealed ArT cleaves 50 additional transcripts, which shows that the endoribonuclease activity of GhoS has been broadened as it was converted to ArT. Furthermore, we evolved an antitoxin for the new toxin ArT from two unrelated antitoxin templates, the protein-based antitoxin MqsA and RNA-based antitoxin ToxI, and showed that the evolved MqsA and ToxI variants are able to counteract the toxicity of ArT. In addition, the de novo TA system was found to increase persistence, a phenotype commonly associated with TA systems. Therefore, toxins and antitoxins from disparate systems can be interconverted.

Bacterial persister cell formation and dormancy.

Bacterial cells may escape the effects of antibiotics without undergoing genetic change; these cells are known as persisters. Unlike resistant cells that grow in the presence of antibiotics, persister cells do not grow in the presence of antibiotics. These persister cells are a small fraction of exponentially growing cells (due to carryover from the inoculum) but become a significant fraction in the stationary phase and in biofilms (up to 1%). Critically, persister cells may be a major cause of chronic infections. The mechanism of persister cell formation is not well understood, and even the metabolic state of these cells is debated. Here, we review studies relevant to the formation of persister cells and their metabolic state and conclude that the best model for persister cells is still dormancy, with the latest mechanistic studies shedding light on how cells reach this dormant state.

Arrested protein synthesis increases persister-like cell formation.

Biofilms are associated with a wide variety of bacterial infections and pose a serious problem in clinical medicine due to their inherent resilience to antibiotic treatment. Within biofilms, persister cells comprise a small bacterial subpopulation that exhibits multidrug tolerance to antibiotics without undergoing genetic change. The low frequency of persister cell formation makes it difficult to isolate and study persisters, and bacterial persistence is often attributed to a quiescent metabolic state induced by toxins that are regulated through toxin-antitoxin systems. Here we mimic toxins via chemical pretreatments to induce high levels of persistence (10 to 100%) from an initial population of 0.01%. Pretreatment of Escherichia coli with (i) rifampin, which halts transcription, (ii) tetracycline, which halts translation, and (iii) carbonyl cyanide m-chlorophenylhydrazone, which halts ATP synthesis, all increased persistence dramatically. Using these compounds, we demonstrate that bacterial persistence results from halted protein synthesis and from environmental cues.