A study of innate immune kinetics reveals a role for a chloride transporter in a virulent Francisella tularensis type B strain

Abstract Tularemia is a zoonotic disease of global proportions. Francisella tularensis subspecies tularensis (type A) and holarctica (type B) cause disease in healthy humans, with type A infections resulting in higher mortality. Repeated passage of a type B strain in the mid‐20th century generated the Live Vaccine Strain (LVS). LVS remains unlicensed, does not protect against high inhalational doses of type A, and its exact mechanisms of attenuation are poorly understood. Recent data suggest that live attenuated vaccines derived from type B may cross‐protect against type A. However, there is a dearth of knowledge regarding virulent type B pathogenesis and its capacity to stimulate the host's innate immune response. We therefore sought to increase our understanding of virulent type B in vitro characteristics using strain OR96‐0246 as a model. Adding to our knowledge of innate immune kinetics in macrophages following infection with virulent type B, we observed robust replication of strain OR96‐0246 in murine and human macrophages, reduced expression of pro‐inflammatory cytokine genes from “wild type” type B‐infected macrophages compared to LVS, and delayed macrophage cell death suggesting that virulent type B may suppress macrophage activation. One disruption in LVS is in the gene encoding the chloride transporter ClcA. We investigated the role of ClcA in macrophage infection and observed a replication delay in a clcA mutant. Here, we propose its role in acid tolerance. A greater understanding of LVS attenuation may reveal new mechanisms of pathogenesis and inform strategies toward the development of an improved vaccine against tularemia.

LVS (Public health monograph No. 50. United States-U.S.S.R medical exchange missions, 1956. Public Health Reports [PHS No. 536]). LVS dramatically decreased laboratory-acquired tularemia incidence rates, replacing the heat-killed Foshay vaccine. However, LVS does not protect against high doses of inhalational type A in animals and humans and is thus considered only partially effective (Burke, 1977;Eigelsbach & Downs, 1961;Sawyer et al., 1966). Additionally, LVS is difficult and expensive to manufacture owing to non-immunogenic colony variants that emerge during its production. LVS remains unlicensed after seven decades of research and development, and the characterization of its attenuating mechanisms is incomplete.
Despite these obstacles, LVS is frequently used as a model organism to study the pathogenesis of virulent type A strains, as their genomes possess more than 97% sequence similarity. However, dozens of rearrangements occur in type A compared to type B genomes, confounding direct comparisons of LVS to type A strains. Differences between type A and type B likely stem from insertional elements and regulatory sequences, in addition to (pseudo)gene content and genome organization. In contrast, differences between LVS and virulent type B strains mainly occur within coding sequences (Petrosino et al., 2006). Although the exact progenitor for LVS is unknown, virulent or "wild type" (WT) type B strains are the closest genetic progenitor of LVS and are therefore the most appropriate context for understanding attenuating mechanisms in LVS.
An important yet often overlooked feature distinguishing virulent type B strains from LVS is that WT type B has a case fatality rate of approximately 7%, whereas, no deaths from LVS have been reported (Staples et al., 2006). While extensive epidemiological and phylogeographical data exist for virulent type B strains, they are understudied compared to type A in terms of pathogenesis and the host's innate immune response. Only a handful of studies have measured WT type B replication and infection kinetics within macrophages or other antigen-presenting cells (APCs). Ray et al. demonstrated the ability of strain OR96-0246 to replicate in bone marrow-derived macrophages (BMDMs) from Fischer 344 rats and reported that its replication in rat hepatocytes was as robust as type A strain Schu S4 (Ray et al., 2010). The growth of strain FSC200 in murine BMDMs, bone marrowderived dendritic cells (BMDCs), and in the monocyte cell line J774.2 has also been reported, with both bacterial and host proteomic analyses performed (Bauler et al., 2014;Fabrik et al., 2018;Pávková et al., 2013;Straskova et al., 2012). Lindgren et al. explored differences in iron content between several type A and type B strains and correlated these differences to increased susceptibility of type B strains to H 2 O 2mediated killing (Lindgren et al., 2011). Brown et al. monitored serum from North American cottontail rabbits infected with type B strains OR96-0246 and KY99-3387 and reported a strong humoral response in rabbits that survived the past 14 days (Brown et al., 2015). Many studies of virulent type B strains typically focus on clinical or diagnostic aspects of tularemia but lack mechanistic insights (Adcock et al., 2014;Fritzsch & Splettstoesser, 2010;Johansson et al., 2000Johansson et al., , 2014Stenmark et al., 2003;Versage et al., 2003).
Using the targeted approach of comparative genomics, our lab and others previously aligned the LVS genome to those of WT type B strains and identified 17 genes that are disrupted in LVS but remain intact in WT strains (Petrosino et al., 2006;Rohmer et al., 2006). This list includes proteins involved in ion transport, sugar modification, protein secretion, nutrient acquisition, and intracellular survival within macrophages, along with genes of unknown function.
Only two genes identified in the above studies were explored more extensively. The first encodes a chimeric protein formed by the fusion of two neighboring siderophore genes, fupA, and fupB (FTL_ RS02265). The type A fupA mutant showed reduced virulence in an intradermal mouse infection model; however, the LD 50 was 10-fold lower than LVS, suggesting LVS is attenuated by additional mechanisms (Twine et al., 2005). The second gene encodes a type IV pilus assembly protein, PilA (FTH_RS02055). It was separately shown that pilA deletions in type A and type B strains are attenuated in mice by the subcutaneous route, but only slightly impaired for intracellular replication in vitro (Forslund et al., 2010;Salomonsson et al., 2009).

Salomonsson et al. concluded that since the reintroduction of both
pilA and fupA together restores virulence of LVS in C57BL/6 mice to a level similar to that of WT type B strains, these genes are therefore responsible for LVS attenuation. However, this finding does not preclude the possibility that the reintroduction of other candidate genes could equally restore virulence in LVS. Furthermore, limitations exist in C57BL/6 mice compared to other animal models, as immunization with LVS generally does not protect C57BL/6 mice against type A challenge (Griffin et al., 2014). While the above findings indicate an important role for FupA and PilA, the contribution of the remaining individual gene disruptions to LVS attenuation has yet to be determined, and their roles in virulence are unclear.
We have retargeted the parent Francisella TargeTron™ plasmid for the disruption of all 17 attenuation candidate genes, laying the groundwork for others to study these genes of interest. One such gene is clcA, which encodes a predicted proton:chloride exchange transporter with eleven predicted trans-membrane spanning regions ( Figure A1). Based on studies in the Escherichia coli (E. coli) homolog, Rohmer et al. suggested that ClcA might be important for survival at low pH (Rohmer et al., 2006). We test this hypothesis here and describe the innate immune response kinetics in human and murine macrophages following infection with a virulent type B strain compared to its attenuated counterpart, LVS, and disruption mutant clcA::ltrB Ll . This study adds to a growing body of work focused on increasing our understanding of virulent type B strains, and to our knowledge is the first-ever characterization of a Francisella chloride channel protein.

| Bacterial strains and stock preparation
A list of strains and plasmids used in this study can be found in Table 1. F. tularensis subsp. holarctica strain OR96-0246 NR-648, originally isolated in Oregon in 1996 after a primate facility outbreak, was obtained through BEI Resources, NIAID, NIH and sequenced at Baylor College of Medicine as previously described (Atkins et al., 2015).
Bacterial counts were determined by spot plating serial dilutions on MHII agar +5% fetal bovine serum (FBS) and growing at 37°C with 5% CO 2 for 48 h. Any samples removed from BSL-3 for further analysis were inactivated and confirmed for sterility by plating at 37°C with 5% CO 2 for more than 48 h.

| Mutant and complementation vector construction
Insertion sites for the clcA gene (1419 bp) were predicted using the TargeTron™ algorithm (Sigma-Aldrich) as described in Rodriguez et al. (2009). The 534|535 sense insertion location was chosen (Score: 8.69, E-value: 0.065) and IBS, EBS1d, and EBS2 primers containing complementary sequences to clcA were used to retarget the parent TargeTron™ plasmid pKEK1140, generating pJFP1004 (Table 1). For electroporation of F. tularensis, electro-competent cells were prepared as described in Rodriguez et al. (2009). Prepared cells were transformed with 0.5-1.0 μg TargeTron™ plasmid DNA at 2.5 kV, 600 Ω and 25 μF (Millipore Sigma). Transformed cells were immediately resuspended in 1 ml pre-warmed MHII broth and recovered for 4 h at 30°C with shaking. Cells were plated on MHII agar +5% (v/v) FBS containing 10-50 μg/ml kanamycin (Calbiochem) and grown at 30°C for 4-5 days. Single colonies were streaked onto fresh plates and the presence of the intron was PCR-verified with gene-and intron-specific primers (Table A1). Confirmed insertional mutants were restreaked on MHII agar +5% (v/v) FBS without antibiotic and grown at 37°C for 2 days to cure the plasmid. Plasmid curing was confirmed via PCR (Table A1). To generate the ClcA E. coli complementation plasmid used in acid challenge assays, pFTClcA, WT type B clcA was synthesized into pEZ (Epoch Life Sciences) and inserted into the low-copy plasmid pWSK29 (Wang & Kushner, 1991) at BamHI and XhoI sites to form the pFTClcA complementation plasmid. Expression was confirmed using western blot.

| Acid Challenge
The acid challenge was performed as described previously (Castanie-Cornet et al., 1999;Iyer et al., 2002

| Human and murine macrophage cell lines are permissive for the growth of WT type B
A better understanding of how virulent type B strains behave in macrophages is required to further explore the attenuating mechanisms of LVS. The tularemia mouse model has its limitations for predicting effects in humans, as mice are overly susceptible to F. tularensis infection (Ray et al., 2010). This is demonstrated by the fact that at some doses LVS retains virulence in mice. However, successful Francisella infection generally correlates with its ability to replicate within macrophages, its primary reservoir (Bhatnagar et al., 1994).

| A proton:chloride antiporter mutant shows delayed proliferation inside macrophages
We next sought to determine the individual roles of candidate genes in LVS attenuation identified by our lab and others through comparative genomics (Petrosino et al., 2006;Rohmer et al., 2006).
Upon the prioritization of candidate genes, we focused our efforts on the proton:chloride antiporter encoded by clcA. ClcA is part of the conserved CLC family of chloride channels and transporters with isoforms spanning prokaryotes and eukaryotes (Miller, 2006).
In LVS, a single base-pair deletion in clcA caused a frameshift mutation resulting in an early stop codon in the gene. The outcome was a marked truncation of ClcA (loss of 80% of the full-length protein), which is predicted to have a deleterious impact on protein function (Rohmer et al., 2006). Notably, conserved residues important for protein's function in E. coli occur after truncation, further supporting ClcA is non-functional in LVS ( Figure A3). To study the role of ClcA in  Methods, and Figure A4). Previously adapted for use in Francisella, this system uses a Lactococcus lactis group II intron with an associated ribonucleoprotein complex (RNP) and a native Francisella promoter (Rodriguez et al., 2008(Rodriguez et al., , 2009. ClcA is not predicted to be part of an operon, assuaging concerns of polar effects. To determine whether this disruption is partly responsible for LVS attenuation, we repeated the above macrophage infection assay with the clcA mutant and observed a delay in replication ( Figure 1). In infected human macrophages, no clcA mutant CFU were recovered (or were below the LOD) until 36 HPI, and bacterial counts were significantly lower than those of WT by an average of 2 log CFU/ml. A shorter replication delay was observed in murine macrophages, with 4.7 log CFU/ ml recovered at 18 HPI, and 5.9 log CFU/ml by 36 HPI (Figure 1). An Indeed, recovered clcA mutant CFU were statistically different compared to LVS CFU at multiple time points, but not WT (Table 2). In summary, clcA mutant reached WT levels by 36 HPI in murine macrophages but remained impaired in human macrophages (Table 2).
While the TargeTron™ gene knockout system is specific and permanent, clcA mutant bacteria were collected from macrophages 36 HPI to confirm the continued presence of the gene disruption and that replication was not due to escape mutants ( Figure A4). Meanwhile, no differences were observed between WT and clcA mutant when grown in broth culture ( Figure A5). Thus, a reproducible trend emerged that clcA mutant displays a lag in intracellular growth that is not observed in broth culture, which may have significant consequences in the context of innate immune detection and clearance by macrophages.

| Immune activation profiling highlights differences in WT-infected macrophages compared to attenuated strains
Activated macrophages secrete pro-inflammatory cytokines in response to intracellular pathogens. IL-1β, IL-6, and TNFα are important for acute phase protein production and fever induction, while  (Bauler et al., 2014;Gillette et al., 2014). To explore differences in the ability of WT type B, LVS, and clcA mutant to modulate cytokine gene expression, we employed real-time quantitative reverse transcription PCR (qRT-PCR). Using the comparative Ct method, we compared transcript levels of infected macrophages to those of uninfected macrophages, normalized to the β-actin gene (Figure 2). In human macrophages, gene expression levels for IL-1β, IL-18, and TNFα from WT infections remained low and were never two-fold higher than uninfected macrophages ( infected macrophages was a ten-fold increase at 36 HPI for IL-1β.
For macrophages infected with clcA mutant, modest increases ≤ two-fold were observed at 18 HPI for IL-1β, IL-18, and TNF-a, but rose to two to five times greater than uninfected controls at 36 HPI, coinciding with recoverable CFU. Due to low primer efficiencies, IL-6 and IL-12 were not examined for human macrophages.
A different trend emerged in infected murine macrophages when we compared cytokine expression levels ( Figure 2D-H). Both WT-and LVS-infected macrophages initially showed increased levels of IL-1β, IL-12, and IL-6 compared to uninfected controls at 4 HPI.
However, the expression of these cytokines in WT-infected macro- ( Figure A6A-C). In human macrophages, IL-6 was not detected in WT infection supernatants ( Figure A6D). Low levels of human IL-1β and TNFα were recovered from WT, LVS, and clcA mutant infection supernatants, but were not considered physiologically relevant (<10 pg/ml for all strains, Figure A6E  Human data represents one independent experiment, and no significant differences are reported. Murine data represents two independent experiments each with two technical replicates. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 as determined by two-way ANOVA In contrast, WT type B continues to replicate inside macrophages, suggesting that WT type B may actively suppress the macrophage innate immune response. In support of these findings, lack of murine macrophage cell death 12 HPI with virulent type A strain Schu S4 was previously reported (Bauler et al., 2014). Finally, the decrease in gene expression levels at 36 HPI for all strains may be a result of increasing macrophage cell death at 36 HPI.

| Francisella ClcA functionally complements acid resistance in a clcA-deficient E. coli strain
From the observed delay in replication of clcA mutant in macrophages, we postulated that the primary activity of ClcA occurs early on during the intracellular lifecycle of Francisella. Following phagocytosis, virulent F. tularensis strains readily escape the phagosome and replicate in the host cytosol. A prolonged but similar level of escape is observed between LVS and F. novicida strain U112, which does not cause disease in immunocompetent humans (Chong et al., 2008;Golovliov et al., 2003). However, the environment of the Francisella (Santic et al., 2008). Others still have shown a mere delay in phagosomal escape following treatment with BFA or concanamycin A (Chong et al., 2008). These studies used different strains, cell types, modes of phagosomal uptake, and stringencies for defining phagosomal disruption, all of which could be contributing factors to these contradictory results. We therefore chose to study ClcA in the context of the extensively studied model system of E. coli.
In E. coli, the H + /Cl − antiporter ClcA (previously EriC) functions in the context of extreme acid resistance (XAR). Bacteria counteract low extracellular pH through decarboxylation-linked proton utilization of imported charged amino acids (glutamate and arginine at pH 2.5, and lysine and ornithine at higher pH) (Foster, 2004;Iyer et al., 2003). In E. coli, ClcA acts as an electrical shunt to reverse hyper-polarization of the bacterial membrane that occurs through the proton-consuming decarboxylation process (Foster, 2004). E. coli possesses a second redundant homolog known as clcB (previously mriT), and only when both genes are deleted is a phenotype observed (Iyer et al., 2002). While ClcA and ClcB in E. coli are believed to have different pH optima (Chris Miller, personal correspondence), there is no redundant gene in Francisella. Several F. tularensis type B strains, including OR96-0246, were previously examined for gastric acid resistance, but may not appropriately reflect conditions during a macrophage infection (Adcock et al., 2014). Furthermore, while reductions in viability were observed for cultures exposed to SGF (synthetic gastric fluid, pH 2.5 and 4.0) compared to acidic PBS (pH 2.5), survival was nonetheless higher for WT strains than for attenuated strains such as LVS. Initial experiments to determine acid sensitivity in WT, LVS, and clcA mutant revealed that LVS was indeed sensitive to acid challenge; however, no differences were observed between WT and clcA mutant. Our best explanation is that F I G U R E 3 Cell death of infected macrophages. 96-well plates were seeded with 5 × 10 4 J774A.1 murine (a) or THP-1 human (b) macrophages and infected with either WT type B, attenuated type B strain LVS, or clcA mutant in quadruplicate. At the indicated time points supernatants were transferred to a new plate and cell death was determined by the lactate dehydrogenase assay as described in Materials and Methods. Absorbance values were averaged and converted into percentages. A representative experiment is shown for clcA mutant and human macrophage cell death. Two independent experiments are shown for WT-and LVS-infected murine macrophages. Mean and SEM shown. No significant differences were detected additional protein(s) is (are) disrupted in LVS compared to the clcA mutant that causes LVS to be more sensitive to acid in vitro, but as stated above, these in vitro experiments may not mirror conditions inside macrophages.
We therefore sought to determine whether Francisella ClcA could functionally complement the E. coli MG1655ΔclcAΔclcB double mutant strain under acidic conditions. Overnight cultures of WT MG1655, MG1655ΔclcAΔclcB, and MG1655ΔclcAΔclcB complemented with either inducible plasmid pFTClcA or empty vector pWSK29 were added to acid challenge buffer (ACB, pH 2.5) alone or supplemented with 1.5 mM glutamate, and survival was measured after 2 h. WT E. coli displays only modest survival in ACB alone, but when grown in the presence of glutamate exhibits increased survival ( Figure 4). In contrast, MG1655ΔclcAΔclcB does not recover even with amino acid supplementation, presumably due to a buildup of charge across the membrane that eventually halts the coupled amino acid exchange system (Garcia-Celma et al., 2013). However, upon the complementation of ΔclcAΔclcB with pFTClcA, acid challenge survival was restored by a modest 5%-10%. These levels are similar to those previously shown (10%-30%) for complementation in E. coli (Iyer et al., 2002 ), but may be lower due to less efficient protein localization. In contrast, the ΔclcAΔclcB strain complemented with empty vector control did not recover. While further studies are required to determine if ClcA plays a role specifically within Francisella-containing phagosomes, our results raise the possibility that ClcA contributes to acid survival in Francisella and warrants further investigation.

| DISCUSS ION
To date, LVS remains the current gold standard for protection against F. tularensis in humans (El Sahly et al., 2009;Hornick & Eigelsbach, 1966;McCrumb, 1961). Myriad new experimental killed, subunit, and live attenuated vaccine candidates have emerged within the past decade. However, variability in animal models, complex vaccination routes and regimens, and increased safety risk due to possible reversion are all significant challenges to overcome (Conlan, 2011;Marohn & Barry, 2013;Shen et al., 2004). Until recently, WT type B strains were overlooked for live attenuated vaccine development.
Attempts to find the attenuation "sweet spot" in type A strains, which must balance attenuation and viability, remain unsuccessful.
Such attempts either result in over-attenuation, and therefore loss of protection, or pose a safety risk (reviewed in Jia & Horwitz, 2018).
Exemplifying this point is the progression of one candidate mutant, clpB, which was initially engineered in virulent type A strain Schu S4 and was shown to surpass LVS efficacy against intranasal challenge with more than 50 CFU Schu S4 in mice (providing 80% or greater protection) Rockx-Brouwer et al., 2012;Ryden et al., 2013;Shen et al., 2010;Twine et al., 2012;Wehrly et al., 2009).
A second locus was deleted due to reversion concerns, abolishing all protection. The single clpB gene deletion was subsequently moved into type B strain FSC200, which also out-performed LVS, albeit not to the extent of Schu S4ΔclpB (Golovliov et al.,). Nonetheless, FSC200ΔclpB serves as proof of concept that mutants of type B backgrounds can meet both efficacy and safety standards. Notably, clcA is located just four genes upstream of clpB.
In this study, we used an in vitro model to investigate the innate immune signals that precede a Th1 response in the host (Cole et al., 2006;. Located at primary infection sites, macrophages act as important first responders to initiate an inflammatory response (Gavrilin et al., 2006;Rivera et al., 2016). Together with the TLR-2/MyD88/NF-κB signaling pathway, cytosolic innate immune activation potentiates cleavage of pro-caspase-1 and subsequent secretion of pro-inflammatory cytokines, resulting in pyroptosis (Atianand et al., 2011;Bergsbaken et al., 2009;Cole et al., 2007;Fernandes-Alnemri et al., 2010;Katz et al., 2006;Man et al., 2015;Meunier et al., 2015). This host cell death effectively removes the replicative niche for F. tularensis, mobilizes adaptive immunity, and consequently inhibits bacterial dissemination. However, virulent strains well-adapted to the intracellular niche employ stealth strategies to avoid host detection. This is demonstrated by our observation that WT-infected macrophages show a delay in cell death compared to LVS and clcA mutant in murine macrophages. Thus, immune activation profiles of macrophages reflect the virulence status of Francisella strains.

F I G U R E 4 WT
Francisella type B ClcA can partially restore acid tolerance in an acid-sensitive E. coli mutant. WT E. coli strain MG1655, double mutant ΔclcAΔclcB, or double mutant complemented with pFTClcA or pWSK29 empty vector control (both induced with 2.5 mM IPTG) were plated for survival after incubation for 2 h in acid buffer with 1.5 mM glutamate at pH 2.5. Counts were normalized to PBS input controls, and survival ratios were converted into percentages. For all strains, survival in the absence of glutamate was less than 0.05%. Data represents 4 to 6 independent experiments, with averaged triplicate CFU counts. Mean with SEM shown. *p < 0.05 as determined by the Kruskal-Wallis test with Dunn's post-test to adjust for multiple comparisons have several advantages over, PBMC macrophages (Daigneault et al., 2010;Qin, 2012). Nonetheless, this work should be further validated by studies in primary cells, as well as in rat or murine animal models, which was a limitation of our study. Lastly, one standing weakness of the current study is that we did not perform complementation studies for the clcA mutant in Francisella, but only in E. coli.
The importance of ion channels in Francisella is made clear by their identification in genetic screens, but the roles of these proteins have never been biochemically demonstrated (Meibom & Charbit, 2010). Based on studies in E. coli, Rohmer et al. suggested that ClcA may be important for survival at low pH (Johansson et al., 2000).
As the cytosol is the site of Francisella replication, we hypothesized that the delay we observed for clcA mutant replication was a result of extended time within the Francisella-containing phagosome (FCP), where chloride ions are readily available (Sonawane et al., 2002). It was previously shown that inhibition of phagosomal acidification delays F. tularensis Schu S4 escape into the cytosol (Chong et al., 2008).

Chong et al. conclude that important bacterial components required
for phagosomal escape may be triggered by intravacuolar pH, and the CLC family of chloride channels is activated at a low pH (Garcia-Celma et al., 2013). Specifically, acidification directs conformational changes that activate voltage-gated chloride transporters (Basilio et al., 2014). Our finding that Francisella ClcA can partially rescue an acid-sensitive E. coli strain under low pH conditions offers one potential explanation for conflicting results surrounding the Francisellacontaining phagosome. One hypothesis is that the initial acidification upon phagocytosis is required to activate ClcA, but subsequent ClcA activity would offset acidification before phagosomal escape. In this purely speculative model, ClcA increases resistance to acidic stress or may provide an environmental cue for the expression of virulence factors required for phagosomal escape. Unfortunately, in-depth examination of phagosomal survival and escape was outside the scope of this manuscript, as lack of funding for this project and a change in the laboratory's research focus precluded further experimentation.
The term "nutritional virulence" was recently popularized to describe the ability of a pathogen to adapt its metabolism to use nutrients available in the host (Abu, 2013;Abu Kwaik & Bumann, 2013;Santic & Abu, 2013). More specifically, the acquisition of amino acids through scavenging can be deemed a form of virulence. Ramond et al. report the existence of 11 amino transporters in Francisella belonging to the family of amino acid/polyamine/organocation (APC) transporters (Ramond et al., 2014). Of these, the arginine (ArgP) and glutamate (GadC) transporters were the only two repeatedly identified in four screens and recently shown to be functional amino acid transporters in Francisella novicida strain U112 and F. tularensis subsp. holarctica strain LVS (Kraemer et al., 2009;Maier et al., 2007;Peng & Monack, 2010;Weiss et al., 2007). In F. novicida, gadC was linked to oxidative stress defense and phagosomal escape (Ramond et al., 2014). The authors further demonstrated that Francisella GadC confers equivalent acid resistance in an E. coli gadC mutant (at pH 2.5, no glutamate supplementation). More recently, Ramond et al. showed that disruption of argP in F. novicida results in delayed phagosomal escape and intracellular replication (Ramond et al., 2015).
The same authors also tested argP mutant survival under acid stress, but at pH 4. Lastly, a replication defect was also observed in LVS ΔargP in J774A.1 macrophages, but only after 10-h post-infection.
Thus, these two transporters also clearly play a role during the early intracellular lifecycle of Francisella and may work in tandem with ClcA (Castanie-Cornet et al., 1999). Additional biochemical evidence is needed to determine what roles, if any, these systems play in acid tolerance in the context of macrophage infections.

| CON CLUS IONS
Type B infections are associated with waterborne outbreaks and are on the rise in certain parts of the world including the United States (Appelt et al., 2020;Gehringer et al., 2013;Kilic et al., 2015;Pedati et al., 2015;Tanaka et al., 2008;Yeni et al., 2020

CO N FLI C T O F I NTE R E S T
None declared.