Teaching old drugs new tricks: Addressing resistance in Francisella

Abstract

 

Keywords:

• biofilms
• Francisella
• multidrug resistance
• MDR bacteria
• virulence

This article refers to:

Screen of FDA-approved drug library identifies maprotiline, an antibiofilm and antivirulence compound with QseC sensor-kinase dependent activity in Francisella novicida

Francisella tularensis is a Gram-negative bacterial pathogen and causative agent of the disease tularemia, also known as “rabbit fever.” The Centers for Disease Control and Prevention (CDC) lists the virulent form of F. tularensis as a Tier 1 pathogen based on its ease in aerosolizing and the lethal damage an inhaled infection can cause in humans. Because of its high pathogenicity and aerosolization ease, F. tularensis is classified as a Category A select agent and represents a significant potential threat as a biological weapon. Especially concerning is the emergence of multi-drug resistant (MDR) Francisella strains adding to the already dire public health and safety concerns.^1-3

Discovery of therapeutic strategies that avoid conferring resistance is crucial for any long-term solution to address the problem of MDR bacteria and enhances the motivation for Francisella research. Francisella novicida is a useful model strain for studying virulence, biofilm formation, and drug resistance in the far more virulent strain, F. tularensis. Unlike other bacteria, F. novicida relies on only two intact 2-component systems (TCSs) and two orphaned members for its virulence and resistive strategies, making any of these select members particularly attractive drug targets. Francisella, like all bacteria, utilize TCS signaling pathways to respond to environmental stimuli through concerted communication between a histidine sensor kinase and a response regulator target. If the TCS signaling components malfunction, bacterial defense mechanisms are compromised, rendering them susceptible to environmental stress. When TCS functionality is specifically targeted, using small molecule drugs or biologics for example, even MDR bacteria can once again show vulnerability to the same antibiotics to which they had previously acquired resistance.^4-8 Similar to F. novicida, F. tularensis encodes orphaned TCS components but the identity and number of these differ depending on the substrain. For example, the highly virulent strain F. tularensis Schu S4 encodes QseC, PmrA/QseB, KdpD, and FTT1543 (the conserved homolog of FTN1452 in F. novicida) all as orphans (QseC and KdpD are sensor kinases; PmrA/QseB and FTT1543 are response regulators). TCS signal transduction pathways generally make attractive targets for the development of anti-infective therapeutics since so many cellular processes rely on the downstream activities from both sensor kinases and response regulators.^9-12 The accelerated work over the last 20 years is indicative of their potential for use as therapeutics and has been reviewed elsewhere.^13-15

Recently, efforts have been made to target response regulator proteins in a variety of systems to thwart bacterial resistance and the onset of virulence.^16-19 For example, PmrA/QseB is the lone response regulator orphan in F. novicida and is an important regulator of biofilm formation across the Francisella genus. PmrA/QseB has been shown to be required for virulence and proper expression of Francisella pathogenicity island (FPI) virulence factors,^20,21 many of which themselves are also required for virulence and infectivity.^22,23 PmrA/QseB upregulates the transcription of FPI genes, most notably the levels of intracellular growth locus C (iglC).²⁴ The activation of PmrA/QseB is mediated by phosphorylation from an upstream sensor kinase, the identity of which is still unclear, but this action could be accomplished by KdpD or even multiple kinases.²⁵ A proposed model suggests that Francisella sensor kinases may not necessarily discriminate for a single response regulator partner, but rather phosphorylate their targets more “promiscuously”.²⁵ Regardless, PmrA/QseB appears to form a complex with another virulence factor, MglA,²¹ a regulator of IglC.²⁶ MglA has subsequently been suggested to form a complex with yet another virulence factor, SspA.²⁵ Furthermore, transcription of FPI appears to be controlled by the phosphorylation/activation of PmrA/QseB which then recruits MglA-SspA together with RNA-polymerase to bind target gene promoters and upregulate the virulence pathway.²⁵

However, it has historically been the sensor kinases, not the response regulators, that have attracted the most attention for therapeutic intervention.^27,28 QseC from Francisella is a sensor kinase found in many bacteria including other biothreat pathogens like MDR Salmonella, Coxiella brunettii, and enterohemorrhagic E. coli (EHEC). The operon for QseC in F. novicida does not encode a paired response regulator, unlike KdpD (paired response regulator is KdpE) and FTN1453 (paired response regulator is FTN1452), and is thus considered orphaned. However despite this, QseC has been shown together with PmrA/QseB to regulate biofilm development in F. novicida and is required for virulence.^29,30 QseC in Francisella is homologous to QseC in E. coli, for which structural information is available for two of its individual domain components (the membrane³¹ and cytoplasmic³² domains). Together with QseB, QseC acts as a TCS quorum sensor to control biofilm formation and motility.^33,34 Moreover, QseC is even considered an adrenergic receptor for norepinephrine and LED209 from studies analyzing several Gram-negative systems, which suggested it acts as a principal regulator of virulence in bacteria.^35,36 Based on a notable increase in research interest over the last 10 years, QseC is currently regarded as a prime target for antagonistic drugs to fight Gram-negative bacterial infections.^36-38

Featured in this issue of Virulence, Dean and van Hoek present a fascinating and compelling study from their screen of 420 FDA-approved drugs against F. novicida, and they successfully identified maprotiline, a tetracyclic antidepressant, as having both antivirulence and antibiofilm properties.³⁹ The authors chose to further refine their focus and specifically monitor the drug-induced effects on TCS-dependent pathways. Based on this strategy, maprotiline was importantly found to carry out its activity in a QseC-dependent manner. This is a highly significant finding, as this type of behavior has not previously been reported for maprotiline and builds on the limited repository of molecules capable of this functionality in a Gram-negative bacterium. The authors used mouse and waxworm models to demonstrate that maprotiline treatment can also prolong the survival and even rescue the animal from disease following a F. novicida infection in vivo. Based on their findings, the authors propose that maprotiline acts as a QseC antagonist, revealing it to be a putative sensor kinase target that subsequently leads to repression of iglC. This provides even greater evidence that QseC may work in concert with PmrA/QseB to control both biofilm development and virulence despite being encoded as TCS orphans. The results of this study make a strong case for further investigating maprotiline as a candidate to treat infections from Francisella and other bacterial pathogens. Effectors targeting sensor kinases have been tested in the past, yet few have resulted in FDA-approved anti-infective therapeutics. Here, Dean and van Hoek show that maprotiline offers tremendous potential because of its strong antivirulence activity and its lack of direct inhibition to bacterial growth - thereby rendering it unlikely to confer bacterial resistance. Maprotiline, already approved by the FDA, is an ideal therapeutic prospect. These developments from the van Hoek laboratory are notable and have the power to expand treatments for bacterial infections by combining synergistically with other known antibiotics.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.