Selected molecular pathways that lead to bacterial persistence
The application of the prototype antibiotics penicillin and streptomycin to bacterial infection in the 1940’s marked a historic milestone in medicine and heralded a new era of antimicrobial therapy as the modern standard for infectious disease management. Yet, even in those early days of discovery, scientist Joseph Bigger noted an unexplained phenomenon. Although penicillin treatment of
In this review, we will discuss the known molecular mechanisms that underlie bacterial persistence, the impact of persistence on infectious disease, and the different strategies that are being developed to target persisters in disease. The human toll of pathogen infection has been compounded by the rampant use of antibiotics in the last half-century, leading to the rapid evolution of drug-resistant strains to practically every approved antibiotic. There is a great public health need to identify novel strategies for development of therapeutics to treat pathogen infection. Development of novel therapies that either kill persisters directly or stimulate their reversion to logarithmic growth may effectively reduce disease relapse and shorten the treatment period. It may be the case that a combination therapy comprised of conventional antibiotics that kill replicating pathogens and new drugs that target the metabolically-inactive persisters can also reduce the rate of emergence of antibiotic resistance.
2. Impact of bacterial persistence on infectious disease
Without question, bacterial persistence greatly contributes to the burden of infectious disease, where persisters survive antibiotic treatment to re-infect patients in a frustrating cycle of chronic infection. Many antibiotics have been shown to be only active against dividing bacteria. Persisters are thought to be dormant cells that greatly slow down essential cellular functions that antibiotics generally target, including trancription, translation, cell wall synthesis, and DNA replication. Persisters are found at relatively higher levels in stationary phase compared to logarithmic cultures, consistent with a dormant state. The persistence state has been found in many different bacterial species, including
There is also increasing evidence that persisters mediate drug tolerance in biofilm formation associated with chronic diseases, including endocarditis, gingivitis, and osteomyelitis. Biofilms form a protective environment for persisters, shielding them from the host immune system. (Fig. 2) Biofilms can form readily on in-dwelling devices, such as catheters and prostheses, or on physiological surfaces, such as
3. Molecular mechanisms of bacterial persistence
Despite observance of the persister phenotype since the 1940’s, the genetic regulatory pathways that switch bacteria into the persister phenotype remain poorly understood. Further research on bacterial persistence rapidly declined with the availability of potent antibiotics. Furthermore, there were technical difficulties in obtaining sufficient numbers of persister cells for analysis and a lack of sophisticated and sensitive methods to study rare biological events at single cell resolution. With the recent emergence of antibiotic-resistant bacterial strains, interest in the mechanisms of bacterial persistence has slowly resurged, amid rapid advances in microfluidics and advanced imaging that can be applied to single cell analysis. [9, 10] A list of genes and pathways linked to persistence is listed in Table 1.
In the 1980’s, a genetic screen was performed to select for
The increased level of persister cells has led to the use of
While HipA contributes to persistence in
There is also increasing evidence that bacterial communication via chemical signaling may play a role in establishing persistence. Recently, indole signaling has been implicated in triggering persistence, leading to enhancement of persister formation in
These results suggest that persisters may form through independent parallel mechanisms and do not follow a single linear regulatory pathway. The underlying commonality is that each of these mechanisms leads to a small subset of quiescent or slowly-dividing cells within an otherwise rapidly dividing population. The fact that the great majority of candidate persister genes have been identified in
4. Toxin/Anti-toxin (TA) modules
One common gene family that has been linked to bacterial persistence is the TA loci, which function in adaptation to rapidly changing environmental conditions in many bacteria and Archaea. TA modules are present in the genome of diverse bacteria, with more than 50 modules in
Since the initial mapping of the
Other TA loci, in addition to
Since both toxin and anti-toxin transcripts are co-expressed from a single promoter, the imbalance between the two transcripts is primarily caused by accumulation of specific proteases, such as the Lon protease in
5. Isolation of bulk and single cell persisters
Persisters are a difficult cell population to manipulate, due to their transient nature, low frequency, and mechanistic heterogeneity. A variety of methods have been utilized for persister enrichment and have leveraged
6. Therapeutic strategies that target bacterial persistence
Initially, investigators sought to identify the genetic determinants that mediate persister formation as potential targets to prevent or reverse persistence. Given the number of disparate genes that appear to be involved in persistence, such an approach may prove to be difficult. Nevertheless, identification of bacterial proteins that are essential even in persisters can provide novel targets for drug development. Since persisters exist in a slowed metabolic state, it is likely that changes in environmental parameters can shift pathogen metabolism from persistence to a replicating state. In the last several years, compounds have been identified that have exhibited promise in the switching of persisters into growing cells susceptible to antibiotic killing or in the direct killing of persisters. (Table 2) These strategies can be integrated with current antibiotics regimens to develop novel viable therapies for treatment of infectious disease.
6.1. Metabolite stimulation of aminoglycoside-mediated bacterial killing
A promising strategy for the eradication of persistent bacteria is the combination of an antibiotic that kills actively replicating bacteria with a metabolite that may enhance the susceptibility of the persistent bacteria to antibiotics. An elegant example of this strategy was the addition of metabolites to stimulate cellular metabolism and switch
In both an
6.2. Hyperactivation of ClpP protease kills persisters
Modulation of target protein function that is lethal for microbes is a novel approach for persister cell elimination. In a recent paper, activation of the ClpP protease by the antibiotic acyldepsipeptide 4 (ADEP4) was shown to kill persister cells by degrading over 400 cellular proteins. ADEP exhibits anti-bacterial activity against Gram-positive bacteria
6.3. Drug screening against metabolically-inactive bacteria
Small molecule library screens have been performed to identify compounds that specifically target persisters. A quinolone-derived library was screened against non-multiplying
Another screen was performed to identify compounds from a library composed of 6800 chemicals, based on scaffolds and physicochemical properties, that can effectively kill persisters. Compounds were downselected based on enhanced killing of
6.4. Combination therapy to treat tuberculosis
Combination treatments that target both replicating and persistent bacteria will likely prove to be an effective strategy to combat chronic infections. A good example of this approach is the multiple drug treatment of tuberculosis (TB), a global disease mediated by
Although in a latent state,
To further identify novel inhibitors against dormant mycobacteria, a hypoxic model system was established to screen >600,000 compounds for those that lowered ATP content in a non-replicating
6.5. Bacterial factors that trigger exit from dormancy
Sporulation is another form of persistence in which both pathogen and environmental microbes enter a metabolically-inactive state and become resistant spores under unfavorable conditions. For example, in nutrient-poor environments,
Other protein factors expressed by microbes have been reported to stimulate growth of dormant cells. The environmental microbe,
The rise in antimicrobial drug resistance, alongside the failure of conventional research efforts to discover new antibiotics, will eventually lead to a public health crisis that can drastically curtail our ability to combat infectious disease. Bacterial persistence is an underexplored mechanism by which to develop novel treatments to complement or extend the current repertoire of antibiotics.[66 - 68] Although persisters do not cause overt disease, they act as a pool from which bacteria can emerge from dormancy to cause recurrent infection. Mechanisms of persister formation appear to be highly redundant across different bacterial species, which contributes to the difficulty in identification of universal mechanisms to target and eradicate persistence. To date, the more successful strategies in the lab have been to target cell functions, such as basal energy metabolism and cell wall integrity, that are also essential for persister cell maintenance. Of particular note, addition of metabolites such as mannitol or fructose was shown to potentiate aminoglycoside-mediated killing by generating a proton motive force to stimulate aminoglycoside uptake. Several medicinal chemistry strategies to screen for small molecules effective against persisters have also identified lead targets for potential optimization and rational drug design.
By developing treatments against both persisters and replicating pathogens, it may be possible to shorten antibiotic regimens, especially for deep-seated diseases such as tuberculosis, and reduce relapse rates in patients. Another advantage to combination therapies is potential extension of the useful life of current antibiotics to kill pathogen at a faster rate, and thus slow down the further emergence of antibiotic resistance. Additional strategies to optimize pulse-dosing regimens using multiple antibiotics that include anti-persister drugs may be able to sterilize particularly recalcitrant chronic infections. These types of therapies may be designed for the individual patient as part of an increasingly personalized approach to medicine. Aside from the clinical relevance of bacterial persisters, non-genetic heterogeneity has been found to play important roles in other systems, including susceptibility of cancer cells to treatment, host response to viral infection, and bacterial responses to other stresses. Thus, understanding the mechanisms of cell-to-cell variability will provide insights into the general adaptation of life to variable environments.
The writing of this review was supported by a Defense Threat Reduction Agency (DTRA) grant to EH-G to study molecular mechanisms of bacterial persistence and develop therapeutics stratagies to defeat persistence.
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