Mechanisms of Francisella tularensis Intracellular ...perspectivesinmedicine.cshlp.org/content/3/4/a010314.full.pdf · Mechanisms of Francisella tularensis Intracellular Pathogenesis

Mechanisms of Francisella tularensisIntracellular Pathogenesis

Jean Celli1 and Thomas C. Zahrt2

1Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergyand Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840

2Department of Microbiology and Molecular Genetics, Center for Infectious Disease Research,Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Correspondence: [email protected]

Francisella tularensis is a zoonotic intracellular pathogen and the causative agent of thedebilitating febrile illness tularemia. Although natural infections by F. tularensis are sporadicand generally localized, the low infectious dose, with the ability to be transmitted to humansvia multiple routes and the potential to cause life-threatening infections, has led to concernsthat this bacterium could be used as an agent of bioterror and released intentionally into theenvironment. Recent studies of F. tularensis and other closely related Francisella species havegreatly increased our understanding of mechanisms used by this organism to infect and causedisease within the host. Here, we review the intracellular life cycle of Francisella and high-light key genetic determinants and/or pathways that contribute to the survival and prolifer-ation of this bacterium within host cells.

Francisella are nonmotile, encapsulated, Gram-negative coccobacilli and are facultative in-

tracellular pathogens of humans and many an-imals. The genus consists of three recognizedspecies: Francisella tularensis, Francisella novi-cida, and Francisella philomiragia. F. tularensisis highly infectious and causes a potentially de-bilitating febrile illness known as tularemia. F.novicida and F. philomiragia are rarely pathogen-ic in man and usually only in individuals whoare severely immunocompromised. Although F.novicida has a limited ability to cause disease inhumans, this organism continues to serve as animportant surrogate model to study aspects ofF. tularensis pathogenesis and host response toinfection owing to its reduced biosafety require-

ments, the conserved nature of its genome re-lative to pathogenic F. tularensis derivatives, itsapparently similar intracellular life cycle, and itsability to cause a tularemia-like disease in in vivomodel systems of infection.

F. tularensis is transmitted from infectedanimals to humans by multiple routes and cancause disease of varying severities depending onthe portal of entry, infectious dose, and subspe-cies (biovar) of the infecting strain. Person-to-person transmission of F. tularensis has not yetbeen reported. F. tularensis subspecies tularen-sis is the most infectious biovar (ID50 , 10 cfu)and is responsible for most cases of tularemia inNorth America (Saslaw et al. 1961a). This sub-species causes the most severe disease symptoms

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and has mortality rates approaching 60% if un-treated (Saslaw et al. 1961a,b; Dienst 1963).Type A strain Schu S4 is the most commonly stud-ied isolate from this subspecies. F. tularensissubspecies holarctica has an infectious dose,103 cfu and is the primary cause of tularemiain Europe and other regions in the NorthernHemisphere. Infections by this subspecies aregenerally associated with milder disease symp-toms and are rarely fatal. The live vaccine strain(LVS) is an attenuated isolate derived from thissubspecies and was developed in the formerSoviet Union. However, it is not licensed foruse in the United States. The remaining biovar,F. tularensis subspecies mediastica, only occa-sionally causes disease in humans.

Infection by F. tularensis occurs primarilyafter inadvertent exposure to infected wildlifespecies, most frequently rodents, hares, and rab-bits. Transmission to humans occurs via directcontact, through arthropod or insect vectors,by ingestion of contaminated material(s), orby inhalation of aerosolized organisms. Regard-less of the entry route, F. tularensis can dissem-inate from the initial infection site to the lungswhere it can cause respiratory tularemia, themost severe form of the disease. The low infec-tious dose, with the ability to be transmitted tohumans via multiple routes, and potential tocause life-threatening illness has resulted in thedesignation of F. tularensis by the United StatesCenters for Infectious Disease Control and Pre-vention as a Category A Select Agent with po-tential to be weaponized and/or intentionallyreleased into the environment. These character-istics have resulted in a renewed interest in thestudy of Francisella, including characterizationof the F. tularensis life cycle, and identification ofbacterial and/or host determinants importantfor aspects of its pathogenesis.

OVERVIEW OF THE Francisella LIFE CYCLE

Although Francisella shows an extracellularphase during bacteriemia in mice (Forestal etal. 2007), survival and replication within hostcells is thought to be a key aspect of its life cycle.This is exemplified by the ability of variousstrains of F. tularensis subsp. tularensis and ho-

larctica and of F. novicida to enter, survive, andproliferate within a variety of host-cell types,including macrophages, dendritic cells, poly-morphonuclear neutrophils, hepatocytes, en-dothelial, and type II alveolar lung epithelialcells (Oyston et al. 2004; McCaffrey and Allen2006; Hall et al. 2007, 2008). Because intracellu-lar proliferation is essential to Francisella viru-lence, much research has focused on under-standing and characterizing specific steps inthe intracellular cycle of this bacterium. It hasbecome clear that Francisella survival and pro-liferation strategies rely on physical escape fromits original phagosome and replication in thehost-cell cytosol (Fig. 1), making this bacteriuma typical cytosol-dwelling pathogen.

Francisella ENTRY INTO MAMMALIAN CELLS

Although entry into nonphagocytic cells re-mains to be further defined, phagocytosis ofFrancisella by macrophages has been extensivelystudied and involves the engagement of differentphagocytic receptors depending on the bacte-rium’s opsonization conditions. The mannosereceptor (MR) plays a significant role in nonop-sonic uptake of F. novicida and F. tularensisstrains by either human monocyte-derived mac-rophages (MDMs), murine bone marrow-de-rived macrophages (BMMs), or J774A.1 macro-phage-like cells (Balagopal et al. 2006; Schulertand Allen 2006; Geier and Celli 2011). Addition-al, yet-to-be-identified receptors are also likelyengaged by nonopsonized Francisella. Serumopsonization, which markedly enhances Franci-sella uptake (Clemens et al. 2004, 2005; Balago-pal et al. 2006; Schulert and Allen 2006; Geierand Celli 2011), mostly redirects the bacteriumto the complement receptor CR3 in human andmurine macrophages, human neutrophils,and dendritic cells (Balagopal et al. 2006; BenNasr et al. 2006; Schulert and Allen 2006; Barkeret al. 2009a). The scavenger receptor A (SR-A)(Pierini 2006; Geier and Celli 2011), Fcg recep-tors (Balagopal et al. 2006), nucleolin (Barel et al.2008), and the lung surfactant protein A (SP-A)(Balagopal et al. 2006) have also been implicatedtovarious degrees in uptake of serum-opsonizedFrancisella by murine or human macrophages.

J. Celli and T.C. Zahrt

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Conversely, Fcg receptors are the main pha-gocytic receptors engaged during uptake of an-tibody-opsonized Francisella, which is also en-hanced compared with nonopsonic conditions(Balagopal et al. 2006; Geier and Celli 2011).Opsonophagocytosis of microbes is generallyas-sociated with enhanced uptake, as is the case forFrancisella. This suggests that this pathogen ex-poses a limited numberof ligands to nonopsonicphagocytic receptors, possibly providing thebacterium with means to limit uptake by bacter-icidal phagocytes. Additionally, such bacterialligands may engage receptors that trigger lessbactericidal pathways, thereby ensuring betterintracellular survival. Consistent with this hy-pothesis, intracellular proliferation of nonop-sonic SchuS4 in murine BMMs is superior tothatofopsonized bacteria (GeierandCelli2011).

THE Francisella-CONTAINING PHAGOSOME

Following uptake, Francisella resides within aphagosome, the Francisella-containing phago-

some (FCP), an initial vacuolar compartmentalong the endocytic degradative pathway thatis normally subjected to progressive matura-tion into a bactericidal phagolysosome. New-ly formed FCPs sequentially acquire markersof early endosomes and late endosomes, suchas EEA-1, CD63, LAMP-1, LAMP-2, and Rab7(Clemens et al. 2004, 2009; Santic et al. 2005a,2008; Checroun et al. 2006; Chong et al. 2008;Wehrly et al. 2009), indicative of a normal mat-uration process. Yet, such interactions do notproceed toward fusion with lysosomes, asFCPs do not seem to accumulate lysosomal lu-minal hydrolases, such as cathepsin D, or lyso-somal tracers (Anthony et al. 1991; Clemenset al. 2004; Santic et al. 2005b; Bonquist et al.2008) and bacteria physically disrupt the phag-osomal membrane and escape into the host-cellcytosol (see below). Another important featureof phagosomal maturation is the progressiveacidification of the phagosomal lumen, via therecruitment of the vacuolar ATPase (v-ATPase),which is both a requirement and a consequence

Cytosolicreplication

Lys

LEEE

EE

LE

LysFCP

Phagosomalescape

Phagocytosis

Reinfection

Cell death

Figure 1. Model of the Francisella intracellular cycle depicting stages that are common to murine and humanphagocytes. Upon phagocytosis, bacteria reside in an early phagosome (FCP) that interacts with early (EE) andlate (LE) endocytic compartments but not lysosomes (Lys). Bacteria rapidly disrupt the FCP membrane andreach the cytosol where they undergo extensive replication, a process followed by cell death, bacterial release,and subsequent infection.

Francisella tularensis Pathogenesis

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of phagosomal maturation (Huynh and Grin-stein 2007). Whether the FCP becomes acidifiedor not before disruption remains contentious.Some studies have shown that FCPs containingeither LVS, Schu S4, or a F. tularensis clinicalisolate resist acidification and acquire limitedamounts of v-ATPase (Clemens et al. 2004; Bon-quist et al. 2008; Cremer et al. 2009), whereasothers report that FCPs containing either F.novicida strain U112 or SchuS4 become acid-ified and acquire the v-ATPase proton pump be-fore phagosomal disruption (Chong et al. 2008;Santic et al. 2008). Additional controversy re-sides in the fact that studies that found thatFCPs become acidified show a role for acidifica-tion in optimal phagosomal escape (Chong et al.2008; Santic et al. 2008), whereas a study inwhich FCPs were found to resist acidificationdid not observe any effect of acidification in-hibitors (Clemens et al. 2009). It is noteworthythat acidified FCPs are observed using nonop-sonic infection conditions, whereas nonacidi-fied FCPs are generated using serum-opsonizedFrancisella, suggesting that the mode of uptakemay affect acidification of the FCP and accountfor discrepancy in the literature. Although FCPintraluminal pH remains to be measured underdifferent opsonization conditions, these studiesnonetheless suggest that physicochemical cueswithin the FCP contribute to Francisella intra-cellular fate. This reinforces the importance ofthe FCP in the Francisella intracellular cycle,in agreement with the fact that it constitutesthe site of intracellular induction of virulencegenes encoded within the Francisella pathoge-nicity island (FPI), a genetic loci that has beenpredicted to encode a type VI secretion system(Nano et al. 2004; Chong et al. 2008; Wehrly et al.2009).

REACTIVE OXYGEN SPECIES ANDINHIBITION OF NADPH OXIDASEACTIVATION

A major bactericidal function of phagosomesis production of reactive oxygen species (ROS)during the oxidative burst, a process mediatedby recruitment and assembly of a multiproteincomplex called the NADPH oxidase onto the

phagosomal membrane. This enzyme mediatesthe conversion of molecular oxygen to superox-ide (O2

2) anions, an ROS that can be subse-quently converted to other reactive speciesincluding hydrogen peroxide (H2O2), hy-pochlorous acid (bleach), and peroxynitrite(ONOO2) within certain cell types or undercertain conditions (Zahrt and Deretic 2002).The NADPH oxidase complex consists of twointegral membrane proteins (gp91phox andp22phox) that together form flavocytochromeb558, and various soluble components that aretranslocated to the phagosomal membrane onactivation. Flavocytochrome b558 contains theredox center of NADPH oxidase and is thedocking site for cytosolic subunits of the en-zyme complex including p47phox, p40phox, andp67phox. Translocation of these determinants toflavocytochrome b558 requires phosphorylationof p47phox and p40phox; activation of NADPHoxidase also requires association with a smallG protein such as Rac1 or Rac2 (Babior 2004).

To ensure their survival, many bacterialpathogens have evolved strategies to resist and/or interfere with ROS generation. For Franci-sella, these survival strategies have been best elu-cidated in human polymorphonuclear neutro-phils (PMNs) (McCaffrey and Allen 2006; Allenand McCaffrey 2007; Buchan et al. 2009; Schu-lert et al. 2009; McCaffrey et al. 2010; Mohapatraet al. 2010), a cell type normally encounteredby the bacterium in vivo and a potent producerof NADPH oxidase-generated O2

2 (Allen andMcCaffrey 2007). In these cells, F. tularensisand F. novicida have been shown to actively dis-rupt ROS generation by altering at least twodifferent steps in NADPH oxidase activation:(1) by inhibiting assembly of flavocytochromeb558 in the phagosomal membrane (McCaffreyet al. 2010), and/or (2) by inhibiting the phos-phorylation of p47phox and p40phox (McCaffreyet al. 2010; Mohapatra et al. 2010). Inhibitionof NADPH oxidase assembly by F. tularensisis dependent on opsonization, and is ob-served with strains from both F. tularensis sub-species tularensis and holarctica, including theLVS (McCaffrey et al. 2010; Geier and Celli2011). Interestingly, F. tularensis also possessesthe unique ability to inhibit NADPH oxidase



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activity postcomplex assembly, an event thatlimits subsequent PMN activation by otherstimuli and diminishes ROS production follow-ing phagocytosis of antibody-opsonized F. tu-larensis (McCaffrey et al. 2010). Regardless ofmechanism(s), inhibition of NADPH oxidaseactivity enhances survival of both F. tularensisand F. novicida in this cell type (McCaffrey andAllen 2006; McCaffrey et al. 2010; Mohapatraet al. 2010; Geier and Celli 2011). Similarly,monocytes or MDMs that have been infectedwith F. tularensis or F. novicida also fail to triggera robust and/or productive oxidative burst(Schulert et al. 2009; Mohapatra et al. 2010; Ge-ier and Celli 2011). However, in contrast toPMNs, the inability of monocytes and MDMsto produce ROS following infection is likely ow-ing to Francisella engagement of and/or inter-nalization by receptors not coupled to NADPHoxidase activation (Schulert et al. 2009). Con-sistent with this observation, intracellulargrowth of F. tularensis or F. novicida is similarfollowing ex vivo infection of monocytes and/orMDMs from wild-type mice compared withp47phox2/2 mice (Lindgren et al. 2005; Moha-patra et al. 2010). Furthermore, IgG-opsoniza-tion of F. tularensis results in NADPH oxidase-dependent ROS generation in wild-type mu-rine BMMs, an affect that is not observed inmacrophages lacking gp91phox (Geier and Celli2011).

Although the ability of Francisella to inhibitNADPH oxidase activity in neutrophils in vitrohas been well established, the importance ofneutrophils and/or NADPH oxidase activityin this and other cell types in vivo remains lessclear. Antibody-mediated depletion of neutro-phils from mice, or targeted recruitment of neu-trophils to the lungs using intranasally deliveredrecombinant MIP-2, a neutrophil-chemotacticchemokine, does not affect bacterial burden ortime-to-death kinetics in mice that have beeninfected with F. tularensis compared with un-treated control animals (KuoLee et al. 2011).Similarly, gp91phox2/2 mice show only slightdifferences in time-to-death and overall bacte-rial burden in target organs of infection com-pared with wild-type animals following respira-tory infection with F. tularensis (KuoLee et al.

2011). Thus, although Francisella may possessspecific strategies to inhibit, counteract, or bypassthe production of ROS by neutrophils and otherphagocytes, the importance of the NADPH ox-idase in controlling infection by pathogenicbiovars of Francisella appears limited in murinetularemia.

Several bacterial determinants have beenimplicated in the resistance of Francisella toROS. For example, Francisella encodes a num-ber of putative acid phosphatases, enzymesthat hydrolyze monoesters at acidic pH, thatmay inhibit the respiratory burst. PurifiedAcpA, the major contributor of acid phospha-tase activity in Francisella, is able to inhibit theoxidative burst in porcine neutrophils that havebeen activated with exogenous stimulants (Reil-ly et al. 1996). Recent evidence suggests thatAcpA is secreted by F. tularensis and F. novicidastrains in vitro (Dai et al. 2011), and is detect-able in the cytosol of macrophages infected withF. novicida shortly after infection (Dai et al.2011). These findings indicate that AcpAwouldbe in a physical location (near the phagosomalmembrane or in the cytosol) to interact withmembrane-bound or soluble components ofthe NADPH oxidase (Dai et al. 2011). Consis-tent with these observations, an F. novicida mu-tant lacking AcpA is partially compromised inits ability to suppress the oxidative burst follow-ing infection of human neutrophils or MDMs(Mohapatra et al. 2010), a phenotype that be-comes more pronounced as additional predict-ed acid phosphatases (acpB, acpC, and hap) aredeleted from the bacterium (Mohapatra et al.2010). Furthermore, loss of AcpA and/orother acid phosphatases reduces the ability ofF. novicida to survive following infection ofthese and other cell types in vitro (Mohapatraet al. 2007, 2008, 2010), and in vivo followingintraperitoneal or intranasal infection of mice(Mohapatra et al. 2007, 2008). The importanceof acid phosphatases in resistance of F. novicidato ROS can also be seen following treatment ofneutrophils and MDMs with reagents that blockassembly and/or function of NADPH oxidase,or in macrophages from p47phox2/2 mice,where the ability of F. novicida DacpA and/orDacpABCH mutants to survive intracellularly is



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improved (Mohapatra et al. 2010). Additionally,ascorbate treatment of murine or human mac-rophages infected with the LVS blocks acidphosphatase production and inhibits intracel-lular growth of the bacterium, a phenotype thatis dependent on production of AcpA (McRaeet al. 2009). Thus, AcpA and other predictedacid phosphatases appear to play an importantrole in NADPH oxidase inhibition and resis-tance to ROS following infection by F. novicida.

It is, however, important to note that AcpAand/or the other acid phosphatases appear toplay little if any role in the inhibition of NADPHoxidase activity or resistance to ROS in phago-cytes that have been infected with human-viru-lent strains of F. tularensis. An acpA mutantgenerated in Schu S4 does not trigger a respira-tory burst and shows similar growth character-istics to the wild-type parent following infectionof human PMNs, even though this bacteriumlacks detectable acid phosphatase activity (Mc-Caffrey et al. 2010). Similarly, deletion of acpA,acpB, or acpC alone or sequentially does notalter growth and/or trafficking characteristicsof Schu S4 in vitro in murine BMMs or humanMDMs, or in vivo following intranasal infectionof mice (Child et al. 2010). Thus, acid phospha-tases may play a different role in F. novicidacompared with F. tularensis, or strains of Fran-cisella that are pathogens of humans may pos-sess/use additional determinants/strategies forROS resistance that are not conserved or func-tional in F. novicida.

Apart from acid phosphatases, other deter-minants have also been implicated in Francisellaresistance to ROS. A number of determinantsinvolved in pyrimidine biosynthesis (carA, carB,and pyrB) mediate resistance to ROS in F. tula-rensis LVS, although it is unclear whether thesegenes act indirectly by simply enhancing thefitness of the bacterium (Schulert et al. 2009).FevR, a transcription factor that regulates ex-pression of �100 genes including those fromthe FPI (Brotcke and Monack 2008; Meibomet al. 2009), is also required for the inhibitionof NADPH oxidase activation and subsequentROS production in PMNs by both F. tularen-sis LVS (Buchan et al. 2009) and F. tularensissubspecies tularensis (McCaffrey et al. 2010).

FTN1133 (FTL0803) encodes a hypotheticalgene product that is required for resistance ofF. novicida and F. tularensis LVS to organic hy-droperoxides (Llewellyn et al. 2011). Mutantslacking this enzyme are attenuated for survivalin macrophages in vitro and show reduced bur-dens in mice following intradermal infection, aphenotype that is partially or completely ab-lated in cells or animals lacking gp91phox (Lle-wellyn et al. 2011). F. tularensis also encodes atleast two superoxide dismutases that mediatethe detoxification of superoxide to H2O2 andO2. sodB, encoding an FeSOD, and sodC, encod-ing CuZn SOD, mediate resistance to paraquat,H2O2, and/or pyrogallol in F. tularensis LVS(Bakshi et al. 2006; Melillo et al. 2009), andare required for virulence of F. tularensis LVS inmacrophages and mice (Bakshi et al. 2006). Theattenuation in virulence observed with thesestrains in vitro and in vivo is attributable toNADPH oxidase, as addition of NADPH oxi-dase inhibitors or utilization of cells or miceunable to make this enzyme complex fail to con-trol infection (Melillo et al. 2009). Finally, F. tu-larensis LVS mutants lacking KatG, a catalase,show increased sensitivity to H2O2 in vitro andare attenuated for virulence in mice in vivo(Lindgrenetal.2007). Interestingly,a similarphe-notype is not observed in strains from subspe-cies tularensis (Lindgren et al. 2007). Thus, Fran-cisella likely uses multiple strategies and a varietyof determinants to mediate resistance to ROS.

Francisella PHAGOSOMAL ESCAPE

Several laboratories have described phagosomalescape by various strains of F. tularensis or F.novicida in either human or murine macro-phages, and other cell types (Golovliov et al.2003; Clemens et al. 2004; Santic et al. 2005a,2008; Checroun et al. 2006; Chong et al. 2008),clearly indicating that this process is conservedand is a hallmark of the Francisella intracellularlife cycle. The study of phagosomal escape ki-netics have nonetheless brought controversy tothe field, as early studies have reported phago-somal disruption and bacterial access to the cy-tosol that range anywhere from 1 h to 8 h post-infection (Golovliov et al. 2003; Clemens et al.



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2004; Santic et al. 2005a; Checroun et al. 2006;McCaffrey and Allen 2006). Such large varia-tions in kinetics are likely attributed to techni-cal differences in the assays and criteria used toassess phagosomal disruption, and also to var-iations in the models studied between laborato-ries where different species and strains of Fran-cisella, and different macrophage models wereexamined. In support of the latter, more recentside-by-side comparisons of phagosomal es-cape kinetics of F. tularensis and F. novicidastrains have highlighted differences in this pro-cess (Chong et al. 2008). For example, morerapid phagosomal escape is observed undernonopsonic conditions (Golovliov et al. 2003;Lindgren et al. 2004; Checroun et al. 2006;Chong et al. 2008; Santic et al. 2008; Barker etal. 2009a; Wehrly et al. 2009; Child et al. 2010;Edwards et al. 2010), whereas slower escape ki-netics occur when opsonic conditions are used(Clemens et al. 2004; McCaffrey and Allen 2006;Schulert et al. 2009). Recent systematic com-parisons of phagosomal escape kinetics by non-opsonic and opsonized SchuS4 in murine mac-rophages have clarified this issue further bydemonstrating that opsonization of Francisellawith either complement or antibodies targetsbacteria to phagocytic pathways that restrictthe extent and timing of phagosomal escape(Geier and Celli 2011). Although the basis forrestricted phagosomal escape on serum opsoni-zation is unclear, that occurring on antibodyopsonization and bacterial targeting to Fcgreceptors depends on the rapid and transientNADPH oxidase-dependent oxidative burst(Geier and Celli 2011) that the bacteria do notseem to be able to prevent, further confirmingthat conditions encountered by phagocytosedFrancisella within the FCP can influence its in-tracellular fate.

Francisella phagosomal escape is requisiteto intracellular proliferation, as exemplified bythe inability of numerous phagosomal escape-deficient mutants to grow within macrophages(Lindgren et al. 2004; Santic et al. 2005b; Bon-quist et al. 2008; Barker et al. 2009b; Wehrly et al.2009; Broms et al. 2012). Given the importantrole of phagosomal escape in the Francisella in-tracellular cycle, much effort has been made to

identify bacterial factors that contribute to thisprocess. Unlike Listeria monocytogenes or Shi-gella flexneri, Francisella does not encode lip-id hydrolases or phospholipases classically in-volved in phagosomal membrane disruption(Larsson et al. 2005). However, F. tularensisdoes appear to express a type VI-like secretionsystem (encoded by the FPI) (Nano et al. 2004),and numerous genes that are expressed fromthis genetic locus or that regulate expressionfrom this locus are important for this phago-somal egress (reviewed in Chong et al. 2008).These include iglC and iglD (Lindgren et al.2004; Santic et al. 2005b; Bonquist et al. 2008;Chong et al. 2008), pdpA (Schmerk et al. 2009),iglI and iglJ (Barker et al. 2009b; McCaffrey et al.2010), vgrG (Barker et al. 2009b; Broms et al.2012), dotU (Broms et al. 2012), mglA (Baronand Nano 1998; Santic et al. 2005b; Bonquistet al. 2008), fevR (Brotcke and Monack 2008;Buchan et al. 2009; Wehrly et al. 2009), andmigR (Buchan et al. 2009). However, other de-terminants not linked to the FPI have also beenimplicated in phagosomal escape by Francisellaincluding the acid phosphatases of F. novicida(AcpA, AcpB, AcpC, and Hap) (Mohapatra etal. 2008), pyrimidine biosynthetic genes (carA,carB, and pyrB) (Schulert et al. 2009), as wellas several genes of unknown function includingFTT1103 (Qin and Mann 2006; Qin et al. 2009)and FTT1676 (Wehrly et al. 2009). Thus, it re-mains unclear whether Francisella use a singlemechanism for escape from the phagosome;further delineation of this process, includingthe genes required, remains a top priority inthe field given its importance for subsequentstages of Francisella survival.

Francisella REPLICATION IN THE CYTOSOL

Besides phagosomal escape, the ability of Fran-cisella to proliferate within the host-cell cytosolis another key aspect of its intracellular life cycle,as exemplified by the avirulence in mice of thelarge number of identified replication-deficientmutants (Brotcke et al. 2006; Tempel et al. 2006;Su et al. 2007; Weiss et al. 2007; Alkhuder et al.2009; Wehrly et al. 2009). Although the cyto-sol may be viewed as permissive for bacterial



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proliferation, compared with a vacuole alongthe endocytic degradative pathway, only cyto-sol-adapted pathogens can readily proliferatewithin this host-cell compartment (O’Riordanand Portnoy 2002), through the expression ofdedicated factors and interactions with hostcomponents that either favor or antagonize bac-terial growth. Because phagosomal escape is aprerequisite to cytosolic replication, identifyingbacterial factors that are specifically required forgrowth in the cytosol (and not for phagosomalescape) via mutagenesis and in vitro and/orin vivo screens has been difficult. In general,identification of specific Francisella cytosolicreplication-defective mutants has required useof more specialized secondary readouts includ-ing those based on electron and/or immuno-fluorescence microscopy. To date, only purinebiosynthetic genes ( purMCD) (Pechous et al.2006, 2008), a g-glutamyl transpeptidase (ggt)(Alkhuder et al. 2009), and several genes ofunknown function including FTT0369c/dipA(Wehrly et al. 2009; Chong et al. 2012),FTT0989 (Brotcke et al. 2006), and ripA (Fulleret al. 2008), have been identified as being spe-cifically required for cytosolic replication byFrancisella. However, it is likely that there re-main a large number of additional gene prod-ucts that also contribute to this process (re-viewed in Chong et al. 2008).

Although Francisella have evolved mecha-nisms to adapt to a cytosolic lifestyle, somehost factors have been identified that either con-tribute or interfere with cytosolic proliferation.Recently, Akimana et al. have identified througha genome-wide RNAi screen in Drosophila mel-anogaster S2Rþ cells the type III PI4-kinasea subunit PI4KC and the ubiquitin-specificpeptidase USP22 as required for cytosolic repli-cation of F. novicida (Akimana et al. 2010).Whether the same host proteins are requiredfor replication of virulent F. tularensis and whattheir function in bacterial replication is remainsto be established. Nonetheless, these findingsargue that this bacterium modulates specifichost pathways associated with lipid signalingand ubiquitin systems to foster replication.

Not surprisingly, innate immune mecha-nisms have been shown to negatively control

intracellular proliferation of Francisella. The cy-tokine interferon (IFN)-g is essential to con-trol primary infections (Leiby et al. 1992; Elkinset al. 1996), and has long been known to re-strict intracellular growth of various Francisellastrains in different host-cell models (Anthonyet al. 1992; Fortier et al. 1992; Polsinelli et al.1994), although the effector mechanisms areunclear. IFN-g control of either LVS or SchuS4 intracellular growth in murine peritoneal ex-udate cells (PECs) seems to depend on genera-tion of nitric oxide by the inducible nitric oxidesynthase (iNOS) (Lindgren et al. 2005, 2007),but not in murine alveolar macrophages (Polsi-nelli et al. 1994), nor in either human blood-derived or murine bone marrow-derived prima-ry macrophages (Edwards et al. 2010). Nonethe-less, independent studies have established thatcytosolic replication rather than phagosomalescape of F. tularensis is the target of IFN-g,as this cytokine does not affect phagosomalescape of either LVS or SchuS4 in eitherJ774A.1 cells and human or murine primarymacrophages (Bonquist et al. 2008; Edwardset al. 2010), but restricts cytosolic proliferation(Edwards et al. 2010). These findings are, how-ever, inconsistent with those of Santic et al.(2005a), who reported that IFN-g activationof human blood-derived macrophages prevent-ed phagosomal escape of F. novicida. Althoughvariations in the experimental designs of thesestudies may account for such contrasting con-clusions, and further work is required to rec-oncile these results and clarify the effectormechanisms induced by IFN-g, these findingsclearly illustrate that phagocytes express spe-cific mechanisms that control cytosolic prolif-eration of Francisella. In addition to the effectof IFN-g on Francisella intracellular growth,alternative activation of macrophages by mastcells via interleukin-4 (IL-4) controls intra-macrophage growth of LVS (Ketavarapu et al.2008). Although increased ATP production,prolonged FCP acidification, and up-regulationof the MR have been invoked in this control(Rodriguez et al. 2010), whether IL-4 affectsFrancisella phagosomal escape and/or cytosol-ic replication remains to be examined in moredepth.



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Francisella AND INNATE IMMUNERECOGNITION

Francisella phagocytosis by macrophages andintracellular trafficking through vacuolar andcytosolic compartments likely subjects the bac-terium to innate immune recognition by vari-ous pattern recognition receptors (PRRs) eitherlocated on the cell surface or endosomes, suchas the Toll-like receptor (TLRs), or in the cyto-solic compartment such as Nod-like receptors(NLRs). TLRs or NLRs typically recognize path-ogen-associated molecular patterns (PAMPs) ordanger-associated molecular patterns (DAMPs)to trigger proinflammatory responses, amongwhich the activation of the inflammasome, acytosolic molecular complex, orchestrates cas-pase-1 activation, processing and release of theproinflammatory cytokines IL-1b and IL-18,and cell death via pyroptosis.

Because of the unique structure of its lipidA moiety (Hajjar et al. 2006), Francisella LPSshows very low endotoxicity and the bacteriumdoes not induce TLR4-mediated signaling. In-stead, LVS significantly stimulates TLR2-me-diated signaling resulting in proinflammatorycytokine production (Katz et al. 2006; Cole etal. 2007), although it is also capable of sub-sequently suppressing TLR-mediated signalingand secretion of proinflammatory cytokines in-duced by other agonists (Telepnev et al. 2003).This ability of Francisella to suppress or dampenproinflammatory responses has been extendedto the virulent subspecies tularensis in humandendritic cells, where infection impairs theiractivation and function (Bosio and Dow 2005;Chase et al. 2009), further supporting the no-tion that Francisella interferes with immune de-tection and responses.

INFLAMMASOME ACTIVATION

Given its cytosolic location, Francisella has be-come a model pathogen to study the function ofinflammasomes, which has also expanded ourunderstanding of how this bacterium may in-terfere with immune recognition. Release of F.novicida in the macrophage cytosol is sensed ina type I IFN-dependent manner (Henry et al.

2007), and activates the absent in melanoma2 (AIM2)-containing inflammasome (Fernan-des-Alnemri et al. 2010; Rathinam et al. 2010;Jones et al. 2011), a type I IFN-inducible com-plex whose activation induces secretion of theproinflammatory cytokines IL1b and pro-IL18and pyroptosis (Mariathasan et al. 2005). AIM2inflammasome activation is potentiated byTLR2 signaling (Jones et al. 2010) and is re-quired in vivo in mice to control bacterialburden by eliminating infected macrophagesthat constitute the Francisella proliferation niche(Mariathasan et al. 2005; Fernandes-Alnemriet al. 2010; Jones et al. 2011). Consistent withAIM2 recognizing cytosolic double-strandedDNA (Rathinam et al. 2010), F. novicida activa-tion of the AIM2 inflammasome is associatedwith bacterial release of DNA through lysisin the cytosol (Jones et al. 2011). These studieshave focused on murine systems and the nonhu-man pathogenic F. novicida species, so an impor-tant question that remains unanswered is wheth-er the conclusions drawn from these studies alsoapply to humans and virulent strains. For exam-ple, recent evidence indicates that infection ofhuman cells with F. novicida, LVS, and the highlyvirulent F. tularensis Schu S4 strains activate theNLRP3 inflammasome in addition to the AIM2inflammasome (Atianand et al. 2011), suggest-ing some functional differences in the innateimmune complexes involved in Francisella rec-ognition between mice and humans. More-over, several studies using F. tularensis subspeciessuggest that infections of BMMs with the LVSstrain (Huang et al. 2010; Ulland et al. 2010), orof human primary macrophages with virulentSchu S4 (Lindemann et al. 2010) do induce littlecytotoxicity compared with F. novicida, raisingthe question of relevance of cytotoxicity in in-fections with virulent tularensis strains.

Given the high adaptation of Francisella tothe cytosol and the potential of proinflamma-tory pathways to restrict its proliferation, thisbacterium has likely evolved mechanisms tocounteract inflammasome activation to preserveits replication niche. In support of this concept,many laboratories have identified hypercy-totoxic mutants of F. novicida (Brotcke et al.2006; Hager et al. 2006; Weiss et al. 2007; Lai



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et al. 2010), LVS (Huang et al. 2010; Ulland et al.2010), and Schu S4 (Lindemann et al. 2010),which typically induced increased inflamma-some-dependent cell death and secretion ofproinflammatory cytokines in either mouse orhuman macrophages. Intriguingly, the hypercy-totoxic phenotype results from the interruptionof genes encoding a variety of proteins, such asthe oligopeptide permease OppB (Brotcke et al.2006), the metallopeptidase PepO (Brotcke et al.2006; Hager et al. 2006), the inner membraneprotein RipA (Huang et al. 2010), the IclR familytranscriptional factor FTT0784 and an unknownprotein FTT0584 (Weiss et al. 2007), and severalproteins involved in biosynthesis of type IV pili,the LPS and the O-antigen polysaccharidic cap-sule (Huang et al. 2010; Ulland et al. 2010),bringing some confusion as to whether Franci-sella specifically suppress inflammasome acti-vation. The hypothesis of Francisella evasion ofinflammasome activation was recently chal-lenged by Peng et al., who showed that manypreviously identified hypercytotoxic mutants ofF. novicida and LVS are deficient in membrane-associated proteins and lyse more extensively inthe cytosol thanwild-type bacteria, consequentlytriggering increased inflammasome activation,secretion of proinflammatory cytokines, and py-roptosis (Peng et al. 2011). Although this studydoes not completely rule out the existence ofFrancisella inflammasome suppression factors,it elegantly argues that most genes identified aspotential suppressors of inflammasome activa-tion do not act specifically and that the hyper-cytotoxic phenotype is in many cases an indirectconsequence of bacterial cell wall fragilizationand enhanced release of PAMPs. Because thisstudy has been performed in F. novicida andLVS, it will also be interesting to verify that theseconclusions also apply to virulent strains.

XENOPHAGIC CAPTURE

Another intracellular innate immune defensemechanism is xenophagy, a selective process ofcapture of intracellular microorganisms withina double-membrane-bound vacuole, the auto-phagosome, for delivery to and degradation intothe lysosomal compartment (Levine et al. 2011).

Francisella cytosolic location makes this patho-gen an ideal substrate for selective autophagicrecognition and capture, a process that sharesmany molecular machineries with nonselective,canonical autophagy. Yet, surprisingly, this bac-terium is able to replicate within murine andhuman macrophages over long periods of time(.18 h) without evidence of a xenophagic re-sponse, suggesting it is eithercapable of avoidingrecognition or inhibiting autophagic processes.A belated autophagic response has nonethelessbeen observed in murine BMMs that enclosesa fraction of cytosolic Francisella into large vac-uoles with autophagic features (Checroun et al.2006), but the role of these vacuoles and theirrelevance is unclear, as they do not form in hu-man macrophages (Akimana et al. 2010; Ed-wards et al. 2010) and may not constitute abona fide stage of the bacterium’s intracellularcycle. Current evidence that Francisella may in-terfere with the autophagy pathway stems fromthe down-regulation of several autophagy-relat-ed genes, such as beclin1, ATG5, ATG12, ATG16L,ATG7, and ATG4a in either Schu S4- or F. novi-cida-infected human monocytes (Butchar et al.2008; Cremer et al. 2009), which suggests thatFrancisella maysuppress an autophagic responseat the gene expression level. Yet, it remains to beshown whether down-regulation of some au-tophagy gene during infection is sufficient toblock this constitutive pathway rapidly enoughto prevent bacteria that have reached the cytosolfrom being targeted. Interestingly, a DdipA(FTT0369c) deletion mutant of SchuS4, whichis deficient in cytosolic replication, is eventuallycaptured and cleared by autophagy following alossof viability in the cytosol (Chong et al. 2012),suggesting that viable Francisella may possessmechanisms that prevent their recognition bythe autophagic machinery. Additionally, wheth-er Francisella infection negatively modulatesnonselective autophagy also needs to be exam-ined to further understand Francisella potentialinterference with this degradative pathway.

CONCLUDING REMARKS

The ability of Francisella to survive and replicatewithin phagocytes and other host cells follow-



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ing infection is a key aspect of its life cycle andrepresents an essential step required by this bac-terium to cause disease within the host. Alter-ations in mechanisms that mediate this ability,including the engagement of appropriate recep-tors, resistance to ROS, escape from the phag-osome, replication within the cytosol, and/or avoidance of innate immune recognition orother defense mechanisms, can lead to alteredsurvival characteristics and potential attenua-tion of F. tularensis in virulence in vitro and/or in vivo. Continued study of the genetic de-terminants contributing to these mechanismswill be essential for the development of thera-peutics or vaccines that are able to specificallyinhibit or prevent disease caused by this bacte-rium.

ACKNOWLEDGMENTS

The authors apologize to those investigators inthe field whose excellent work we were not ableto reference. We are grateful to Anita Mora at theRocky Mountain Laboratory for her assistancewith graphic art. T.C.Z. acknowledges supportfrom the National Institutes of Health, NationalInstitute of Allergy and Infectious Diseases(NIAID) (1R21 AI097597-01A1), and theNIAID’s Regional Center of Excellence for Bio-defense and Emerging Infectious Diseases Re-search Program. Membership within and sup-port from the Region V Great Lakes RegionalCenter of Excellence (National Institutes ofHealth Award 2-U54-AI-057153) is also ac-knowledged. J.C. acknowledges support fromthe Intramural Research Program of the Nation-al Institutes of Health, National Institute of Al-lergy and Infectious Diseases.

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2013; doi: 10.1101/cshperspect.a010314Cold Spring Harb Perspect Med Jean Celli and Thomas C. Zahrt

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Jean Celli and Thomas C. Zahrt

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