Development of a novel sandwich immunoassay based on targeting recombinant Francisella outer membrane protein A for the diagnosis of tularemia.

Introduction: Tularemia, caused by the bacterium Francisella tularensis, poses health risks to humans and can spread through a variety of routes. It has also been classified as a Tier 1 Select agent by the CDC, highlighting its potential as a bioterrorism agent. Moreover, it is difficult to diagnose in a timely fashion, owing to the non-specific nature of tularemia infections. Rapid, sensitive, and accurate detection methods are required to reduce mortality rates. We aimed to develop antibodies directed against the outer membrane protein A of F. tularensis (FopA) for rapid and accurate diagnosis of tularemia. Methods: We used a baculovirus insect cell expression vector system to produce the FopA antigen and generate anti-FopA antibodies through immunization of BALB/c mice. We then employed hybridoma and phage display technologies to screen for antibodies that could recognize unique epitopes on FopA. Result: Two monoclonal antibodies, 6B12 and 3C1, identified through phage display screening specifically bound to recombinant FopA in a dose-dependent manner. The binding affinity of the anti-FopA 6B12 and 3C1 antibodies was observed to have an equilibrium dissociation constant of 1.76 × 10-10 M and 1.32 × 10-9 M, respectively. These antibodies were used to develop a sandwich ELISA system for the diagnosis of tularemia. This assay was found to be highly specific and sensitive, with detection limits ranging from 0.062 ng/mL in PBS to 0.064 ng/mL in skim milk matrices. Discussion: Our findings demonstrate the feasibility of a novel diagnostic approach for detecting F. tularensis based on targeting FopA, as opposed to existing tests that target the bacterial lipopolysaccharide.

TolC and EmrA1 contribute to Francisella novicida multidrug resistance and modulation of host cell death.

Francisella spp. are Gram-negative, facultative intracellular pathogens. Francisella tularensis causes the human disease tularemia and is considered a biological threat agent due to its high infectivity and virulence. A central aspect of Francisella virulence is its ability to dampen host immune responses. We previously identified the outer membrane channel (OMC) protein TolC as a critical F. tularensis virulence factor required for suppression of apoptotic and proinflammatory responses during macrophage infection. TolC functions as part of multidrug efflux systems and the type I secretion pathway that exports bacterial effector proteins. In these systems, TolC forms tripartite complexes together with an inner membrane transporter and periplasmic membrane fusion protein (MFP). To advance understanding of TolC function in Francisella, we analyzed OMC and MFP homologs in Francisella novicida, a widely used model species that causes a tularemia-like disease in mice. In agreement with the previous F. tularensis studies, all three OMCs present in F. novicida contributed to multidrug resistance, but only TolC was important for suppressing macrophage cell death. In addition, we identified the EmrA1 MFP as important for resisting antimicrobial compounds and dampening host cell death. In contrast to results obtained with F. tularensis, the cell death triggered during infection with the F. novicida tolC and emrA1 mutants was dominated by pyroptosis rather than apoptosis. These data expand our understanding of TolC function in Francisella and underscore both conserved and differential aspects of F. novicida and F. tularensis. IMPORTANCE: Francisella tularensis is a Gram-negative intracellular bacterial pathogen and causative agent of tularemia. We previously identified the outer membrane channel protein TolC as contributing to antimicrobial resistance and subversion of host responses by F. tularensis. To advance understanding of TolC function in Francisella and to identify components that might work together with TolC, we took advantage of a transposon mutant library in F. novicida, a model species that causes a tularemia-like disease in mice. Our findings identify TolC and the membrane fusion protein EmrA1 as important for both antimicrobial resistance and suppression of macrophage cell death. This study also revealed differences in cell death pathways triggered by F. novicida versus F. tularensis infection that may relate to differences in virulence.

Oropharyngeal tularemia outbreak linked to drinking contaminated tap water in North-Western Iran.

Tularemia is a widespread bacterial disease caused by Francisella tularensis. Iran is an endemic country for this zoonosis. In this report, we present a 2020 tularemia outbreak in a village in northwestern Iran involving 15 patients with the oropharyngeal form of the disease. This outbreak was probably linked to the consumption of contaminated drinking water.

Structural proteomics guided annotation of vaccine targets and designing of multi-epitopes vaccine to instigate adaptive immune response against Francisella tularensis.

Francisella tularensis can cause severe disease in humans via the respiratory or cutaneous routes and a case fatality ratio of up to 10 % is reported due to lack of proper antibiotic treatment, while F. novicida causes disease in severely immunocompromised individuals. Efforts are needed to develop effective vaccine candidates against Francisella species. Thus, in this study, a systematic computational work frame was used to deeply investigate the whole proteome of Francisella novicida containing 1728 proteins to develop vaccine against F. tularensis and related species. Whole-proteome analysis revealed that four proteins including (A0Q492) (A0Q7Y4), (A0Q4N4), and (A0Q5D9) are the suitable vaccine targets after the removal of homologous, paralogous and prediction of subcellular localization. These proteins were used to predict the T cell, B cell, and HTL epitopes which were joined together through suitable linkers to construct a multi-epitopes vaccine (MEVC). The MEVC was found to be highly immunogenic and non-allergenic while the physiochemical properties revealed the feasible expression and purification. Moreover, the molecular interaction of MEVC with TLR2, molecular simulation, and binding free energy analyses further validated the immune potential of the construct. According to Jcat analysis, the refined sequence demonstrates GC contents of 41.48 % and a CAI value of 1. The in-silico cloning and optimization process ensured compatibility with host codon usage, thereby facilitating efficient expression. Computational immune simulation studies underscored the capacity of MEVC to induce both primary and secondary immune responses. The conservation analysis further revealed that the selected epitopes exhibit 100 % conservation across different species and thus provides wider protection against Francisella.

Avirulence of a spontaneous Francisella tularensis subsp. mediasiatica prmA mutant.

Francisella tularensis, the causative agent of tularemia, is divided into three subspecies. Two of these, subspecies holarctica and tularensis, are highly pathogenic to humans and consequently relatively well studied. The third subspecies, mediasiatica, is rarely isolated and remains poorly studied. It is distributed in the sparsely populated regions of Central Asia and Siberia. Curently this subspecies is not known to have been responsible for human infections in spite of its high virulence in laboratory animals. Subspecies mediasiatica is currently divided into three subgroups-MI, present in Central Asia, MII, present in southern Siberia, and MIII represented by a unique strain, 60(B)57, isolated in Uzbekistan in 1960. We describe here the unexpected observation that MIII strain 60(B)57 is avirulent and immunogenic. We observed that infection with this strain protected mice from challenge 21 days later with a virulent subsp. mediasiatica strain. With an increase of this interval, the protection for mice was significantly reduced. In contrast, guinea pigs were protected from challenge with strains of the subspecies holarctica and mediasiatica (but not subsp. tularensis) 90 days after infection with 60(B)57. We performed genome assembly based on whole genome sequencing data obtained using the Nanopore MinION for strain 60(B)57 and two subsp. mediasiatica strains representing the Central Asian MI and Siberian MII phylogenetic subgroups. The prmA gene is truncated due to a nonsense mutation in strain 60(B)57. The deletion of gene prmA has previously been shown to induce a loss of virulence in Francisella novicida the closest model organism suggesting that the observed mutation might the cause of the avirulence of strain 60(B)57.

The geographic distribution, and the biotic and abiotic predictors of select zoonotic pathogen detections in Canadian polar bears.

Increasing Arctic temperatures are facilitating the northward expansion of more southerly hosts, vectors, and pathogens, exposing naïve populations to pathogens not typical at northern latitudes. To understand such rapidly changing host-pathogen dynamics, we need sensitive and robust surveillance tools. Here, we use a novel multiplexed magnetic-capture and droplet digital PCR (ddPCR) tool to assess a sentinel Arctic species, the polar bear (Ursus maritimus; n = 68), for the presence of five zoonotic pathogens (Erysipelothrix rhusiopathiae, Francisella tularensis, Mycobacterium tuberculosis complex, Toxoplasma gondii and Trichinella spp.), and observe associations between pathogen presence and biotic and abiotic predictors. We made two novel detections: the first detection of a Mycobacterium tuberculosis complex member in Arctic wildlife and the first of E. rhusiopathiae in a polar bear. We found a prevalence of 37% for E. rhusiopathiae, 16% for F. tularensis, 29% for Mycobacterium tuberculosis complex, 18% for T. gondii, and 75% for Trichinella spp. We also identify associations with bear age (Trichinella spp.), harvest season (F. tularensis and MTBC), and human settlements (E. rhusiopathiae, F. tularensis, MTBC, and Trichinella spp.). We demonstrate that monitoring a sentinel species, the polar bear, could be a powerful tool in disease surveillance and highlight the need to better characterize pathogen distributions and diversity in the Arctic.

Insights into DNA-binding motifs and mechanisms of Francisella tularensis novicida two-component system response regulator proteins QseB, KdpE, and BfpR.

Two component system bacterial response regulators are typically DNA-binding proteins which enable the genetic regulation of many adaptive bacterial behaviors. Despite structural similarity across response regulator families, there is a diverse array of DNA-binding mechanisms. Bacteria usually encode several dozen two-component system response regulators, but Francisella tularensis only encodes three. Due to their simplified response regulatory network, Francisella species are a model for studying the role of response regulator proteins in virulence. Here, we show that Francisella response regulators QseB, KdpE, and BfpR all utilize different DNA-binding mechanisms. Our evidence suggests that QseB follows a simple mechanism whereby it binds a single inverted repeat sequence with a higher affinity upon phosphorylation. This behavior is independent of whether QseB is a positive or negative regulator of the gene as demonstrated by qseB and priM promoter sequences, respectively. Similarly, KdpE binds DNA more tightly upon phosphorylation, but also exhibits a cooperative binding isotherm. While we propose a KdpE binding site, it is possible that KdpE has a complex DNA-binding mechanism potentially involving multiple copies of KdpE being recruited to a promoter region. Finally, we show that BfpR appears to bind a region of its own promoter sequence with a lower affinity upon phosphorylation. Further structural and enzymatic work will need to be performed to deconvolute the KdpE and BfpR binding mechanisms.

The Epidemiological Investigation of Yersinia pestis, Francisella tularensis, and Arenavirus Infections in Small Mammals in Northwestern Iran.

Background: The control and prevention of rodent-borne diseases are mainly based on our knowledge of ecology and the infectious status of their reservoir hosts. This study aimed to evaluate the prevalence of Francisella tularensis, Yersinia pestis, and arenavirus infections in small mammals and to assess the potential of disease occurrence in East Azerbaijan, northwest of Iran, in 2017 and 2018. Methods: Spleen and lung samples were obtained from all trapped small mammals. The real-time quantitative PCR (qPCR) method was used to detect nucleic acid sequences of F. tularensis, Y. pestis, and arenaviruses. Serum samples were tested for antibodies indicating the host response to F. tularensis and Y. pestis infections using the standard tube agglutination test and enzyme-linked immunosorbent assay (ELISA), respectively. Results: A total of 205 rodents, four Eulipotyphla, and one carnivore were captured. The most common rodent species captured (123 of 205 rodents, 60%) belonged to the genus Meriones (mainly Persian jird, Meriones persicus). In total, 317 fleas were removed from trapped animals. Flea species belonged to Xenopsylla buxtoni, Xenopsylla nuttalli, Stenoponia tripectinata, Paraceras melis, Ctenophthalmus rettigi smiti, Rhadinopsylla bivirgis, Paradoxopsyllus grenieri, and Nosopsyllus iranus. Using the qPCR tests, five spleen samples from M. persicus were positive for F. tularensis. The qPCR tests were negative for the detection of Y. pestis and arenaviruses. Finally, all serum samples tested were negative for antibodies against Y. pestis and F. tularensis. Conclusions: F. tularensis was the only zoonotic agent detected in rodents captured in East Azerbaijan. However, the diversity of trapped rodents and fleas provides the potential for the spread of various rodent-borne viral and bacterial diseases in the studied areas.

Polyaminated, acetylated and stop codon readthrough of recombinant Francisella tularensis universal stress protein in Escherichia coli.

Recombinant Francisella tularensis universal stress protein with a C-terminal histidine-tag (rUsp/His6) was expressed in Escherichia coli. Endogenous F. tularensis Usp has a predicted molecular mass of 30 kDa, but rUsp/His6 had an apparent molecular weight of 33 kDa based on Western blot analyses. To determine the source of the higher molecular weight for rUsp/His6, post translational modifications were examined. Tryptic peptides of purified rUsp/His6 were subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) and fragmentation spectra were searched for acetylated lysines and polyaminated glutamines. Of the 24 lysines in rUsp/His6, 10 were acetylated (K63, K68, K72, K129, K175, K201, K208, K212, K233, and K238) and three of the four glutamines had putrescine, spermidine and spermine adducts (Q55, Q60 and Q267). The level of post-translational modification was substoichiometric, eliminating the possibility that these modifications were the sole contributor to the 3 kDa extra mass of rUsp/His6. LC-MS/MS revealed that stop codon readthrough had occurred resulting in the unexpected addition of 20 extra amino acids at the C-terminus of rUsp/His6, after the histidine tag. Further, the finding of polyaminated glutamines in rUsp/His6 indicated that E. coli is capable of transglutaminase activity.

Dual proteomics of infected macrophages reveal bacterial and host players involved in the Francisella intracellular life cycle and cell to cell dissemination by merocytophagy.

Bacterial pathogens adapt and replicate within host cells, while host cells develop mechanisms to eliminate them. Using a dual proteomic approach, we characterized the intra-macrophage proteome of the facultative intracellular pathogen, Francisella novicida. More than 900 Francisella proteins were identified in infected macrophages after a 10-h infection. Biotin biosynthesis-related proteins were upregulated, emphasizing the role of biotin-associated genes in Francisella replication. Conversely, proteins encoded by the Francisella pathogenicity island (FPI) were downregulated, supporting the importance of the F. tularensis Type VI Secretion System for vacuole escape, not cytosolic replication. In the host cell, over 300 proteins showed differential expression among the 6200 identified during infection. The most upregulated host protein was cis-aconitate decarboxylase IRG1, known for itaconate production with antimicrobial properties in Francisella. Surprisingly, disrupting IRG1 expression did not impact Francisella's intracellular life cycle, suggesting redundancy with other immune proteins or inclusion in larger complexes. Over-representation analysis highlighted cell-cell contact and actin polymerization in macrophage deregulated proteins. Using flow cytometry and live cell imaging, we demonstrated that merocytophagy involves diverse cell-to-cell contacts and actin polymerization-dependent processes. These findings lay the groundwork for further exploration of merocytophagy and its molecular mechanisms in future research.Data are available via ProteomeXchange with identifier PXD035145.

Identification of an N-terminal tag (580N) that improves the biosynthesis of fluorescent proteins in Francisella tularensis and other Gram-negative bacteria.

Utilization of fluorescent proteins is widespread for the study of microbial pathogenesis and host-pathogen interactions. Here, we discovered that linkage of the 36 N-terminal amino acids of FTL_0580 (a hypothetical protein of Francisella tularensis) to fluorescent proteins increases the fluorescence emission of bacteria that express these recombinant fusions. This N-terminal peptide will be referred to as 580N. Western blotting revealed that the linkage of 580N to Emerald Green Fluorescent Protein (EmGFP) in F. tularensis markedly improved detection of this protein. We therefore hypothesized that transcripts containing 580N may be translated more efficiently than those lacking the coding sequence for this leader peptide. In support, expression of emGFPFt that had been codon-optimized for F. tularensis, yielded significantly enhanced fluorescence than its non-optimized counterpart. Furthermore, fusing emGFP with coding sequence for a small N-terminal peptide (Serine-Lysine-Isoleucine-Lysine), which had previously been shown to inhibit ribosomal stalling, produced robust fluorescence when expressed in F. tularensis. These findings support the interpretation that 580N enhances the translation efficiency of fluorescent proteins in F. tularensis. Interestingly, expression of non-optimized 580N-emGFP produced greater fluorescence intensity than any other construct. Structural predictions suggested that RNA secondary structure also may be influencing translation efficiency. When expressed in Escherichia coli and Klebsiella pneumoniae bacteria, 580N-emGFP produced increased green fluorescence compared to untagged emGFP (neither allele was codon optimized for these bacteria). In conclusion, fusing the coding sequence for the 580N leader peptide to recombinant genes might serve as an economical alternative to codon optimization for enhancing protein expression in bacteria.

Mutation of wbtJ, a N-formyltransferase involved in O-antigen synthesis, results in biofilm formation, phase variation and attenuation in Francisella tularensis.

Two clinically important subspecies, Francisella tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B) are responsible for most tularaemia cases, but these isolates typically form a weak biofilm under in vitro conditions. Phase variation of the F. tularensis lipopolysaccharide (LPS) has been reported in these subspecies, but the role of variation is unclear as LPS is crucial for virulence. We previously demonstrated that a subpopulation of LPS variants can constitutively form a robust biofilm in vitro, but it is unclear whether virulence was affected. In this study, we show that biofilm-forming variants of both fully virulent F. tularensis subspecies were highly attenuated in the murine tularaemia model by multiple challenge routes. Genomic sequencing was performed on these strains, which revealed that all biofilm-forming variants contained a lesion within the wbtJ gene, a formyltransferase involved in O-antigen synthesis. A ΔwbtJ deletion mutant recapitulated the biofilm, O-antigen and virulence phenotypes observed in natural variants and could be rescued through complementation with a functional wbtJ gene. Since the spontaneously derived biofilm-forming isolates in this study were a subpopulation of natural variants, reversion events to the wbtJ gene were detected that eliminated the phenotypes associated with biofilm variants and restored virulence. These results demonstrate a role for WbtJ in biofilm formation, LPS variation and virulence of F. tularensis.

The rLVS ΔcapB/iglABC vaccine provides potent protection in Fischer rats against inhalational tularemia caused by various virulent Francisella tularensis strains.

Francisella tularensis is one of the several biothreat agents for which a licensed vaccine is needed. To ensure vaccine protection is achieved across a range of virulent F. tularensis strains, we assembled and characterized a panel of F. tularensis isolates to be utilized as challenge strains. A promising tularemia vaccine candidate is rLVS ΔcapB/iglABC (rLVS), in which the vector is the LVS strain with a deletion in the capB gene and which additionally expresses a fusion protein comprising immunodominant epitopes of proteins IglA, IglB, and IglC. Fischer rats were immunized subcutaneously 1-3 times at 3-week intervals with rLVS at various doses. The rats were exposed to a high dose of aerosolized Type A strain Schu S4 (FRAN244), a Type B strain (FRAN255), or a tick derived Type A strain (FRAN254) and monitored for survival. All rLVS vaccination regimens including a single dose of 107 CFU rLVS provided 100% protection against both Type A strains. Against the Type B strain, two doses of 107 CFU rLVS provided 100% protection, and a single dose of 107 CFU provided 87.5% protection. In contrast, all unvaccinated rats succumbed to aerosol challenge with all of the F. tularensis strains. A robust Th1-biased antibody response was induced in all vaccinated rats against all F. tularensis strains. These results demonstrate that rLVS ΔcapB/iglABC provides potent protection against inhalational challenge with either Type A or Type B F. tularensis strains and should be considered for further analysis as a future tularemia vaccine.

Pathogenicity and virulence of Francisella tularensis.

Tularaemia is a zoonotic disease caused by the Gram-negative bacterium, Francisella tularensis. Depending on its entry route into the organism, F. tularensis causes different diseases, ranging from life-threatening pneumonia to less severe ulceroglandular tularaemia. Various strains with different geographical distributions exhibit different levels of virulence. F. tularensis is an intracellular bacterium that replicates primarily in the cytosol of the phagocytes. The main virulence attribute of F. tularensis is the type 6 secretion system (T6SS) and its effectors that promote escape from the phagosome. In addition, F. tularensis has evolved a peculiar envelope that allows it to escape detection by the immune system. In this review, we cover tularaemia, different Francisella strains, and their pathogenicity. We particularly emphasize the intracellular life cycle, associated virulence factors, and metabolic adaptations. Finally, we present how F. tularensis largely escapes immune detection to be one of the most infectious and lethal bacterial pathogens.

Positive Francisella tularensis meningitis outcome despite delayed identification: a case report.

Francisella tularensis is a Gram-negative bacteria, that may cause a zoonotic disease, tularemia. Here, we describe a patient case, where a previously healthy young woman in Northern Finland contacted health care because of fever and headache. Due to the symptoms and lack of further diagnostic tools in primary health care, she was transferred to University Hospital (UH) where ampicillin and ceftriaxone was given empirically. A cerebrospinal fluid sample (CSF) was drawn showing small Gram-negative rods that grew on chocolate agar after 2 days of incubation. Matrix-assisted laser-desorption-ionization time of-flight (Maldi-tof) did not provide identification, but the bacteria was interpreted as sensitive to ciprofloxacin and the treatment was changed to ciprofloxacin. During the time the patient was infected, there were several positive tularemia samples found in the area. Therefore, an in house tularemia nucleic acid method (PCR) was used on the bacterial culture. Additionally, 16S rDNA sequencing was performed and these methods identified the bacteria as F. tularensis. Fortunately, the patient recovered completely with ciprofloxacin and was discharged without any complications. Our case underlines the need to understand the limits of specific diagnostic methods, such as Maldi-tof, used in clinical laboratory settings. It also highlights the need of both clinicians and laboratory staff to be aware of the many clinical presentations of tularemia when working in an endemic area.

Three Cases of Tickborne Francisella tularensis Infection, Austria, 2022.

Tularemia is increasing in Austria. We report Francisella tularensis subspecies holarctica isolated from 3 patients who had been bitten by arthropods. Next-generation sequencing showed substantial isolate similarity. Clinicians should consider bloodstream F. tularensis infections for patients with signs/symptoms of ulceroglandular tularemia, and surveillance of potential vectors should be intensified.

Ribosome heterogeneity results in leader sequence-mediated regulation of protein synthesis in Francisella tularensis.

Although ribosomes are generally examined in aggregate, ribosomes can be heterogenous in composition. Evidence is accumulating that changes in ribosome composition may result in altered function, such that ribosome heterogeneity may provide a mechanism to regulate protein synthesis. Ribosome heterogeneity in the human pathogen Francisella tularensis results from incorporation of one of three homologs of bS21, a small ribosomal subunit protein demonstrated to regulate protein synthesis in other bacteria. Loss of one homolog, bS21-2, results in genome-wide post-transcriptional changes in protein abundance. This suggests that bS21-2 can, either directly or indirectly, lead to preferential translation of particular mRNAs. Here, we examine the potential of bS21-2 to function in a leader sequence-dependent manner and to function indirectly, via Hfq. We found that the 5´ untranslated region (UTR) of some bS21-2-responsive genes, including key virulence genes, is sufficient to alter translation in cells lacking bS21-2. We further identify features of a 5´ UTR that allow responsiveness to bS21-2. These include an imperfect Shine-Dalgarno sequence and a particular six nucleotide sequence. Our results are consistent with a model in which a bS21 homolog increases the efficiency of translation initiation through interactions with specific leader sequences. With respect to bS21-2 indirectly regulating translation via the RNA-binding protein Hfq, we found that Hfq controls transcript abundance rather than protein synthesis, impacting virulence gene expression via a distinct mechanism. Together, we determined that ribosome composition in F. tularensis regulates translation in a leader sequence-dependent manner, a regulatory mechanism which may be used in other bacteria. IMPORTANCE Ribosome heterogeneity is common in bacteria, and there is mounting evidence that ribosome composition plays a regulatory role in protein synthesis. However, mechanisms of ribosome-driven gene regulation are not well understood. In the human pathogen Francisella tularensis, which encodes multiple homologs for the ribosomal protein bS21, loss of one homolog impacts protein synthesis and virulence. Here, we explore the mechanism behind bS21-mediated changes in protein synthesis, finding that they can be linked to altered translation initiation and are dependent on specific sequences in the leaders of transcripts. Our data support a model in which ribosome composition regulates gene expression through translation, a strategy that may be conserved in diverse organisms with various sources of ribosome heterogeneity.

Human Tularemia Epididymo-Orchitis Caused by Francisella tularensis Subspecies holartica, Austria.

A previously healthy man in Austria had tularemia epididymo-orchitis develop, leading to unilateral orchiectomy. Francisella tularensis subspecies holartica was detected by 16S rRNA gene sequencing analysis of inflamed granulomatous testicular tissue. Clinicians should suspect F. tularensis as a rare etiologic microorganism in epididymo-orchitis patients with relevant risk factors.

Discovery of a glutathione utilization pathway in Francisella that shows functional divergence between environmental and pathogenic species.

Glutathione (GSH) is an abundant metabolite within eukaryotic cells that can act as a signal, a nutrient source, or serve in a redox capacity for intracellular bacterial pathogens. For Francisella, GSH is thought to be a critical in vivo source of cysteine; however, the cellular pathways permitting GSH utilization by Francisella differ between strains and have remained poorly understood. Using genetic screening, we discovered a unique pathway for GSH utilization in Francisella. Whereas prior work suggested GSH catabolism initiates in the periplasm, the pathway we define consists of a major facilitator superfamily (MFS) member that transports intact GSH and a previously unrecognized bacterial cytoplasmic enzyme that catalyzes the first step of GSH degradation. Interestingly, we find that the transporter gene for this pathway is pseudogenized in pathogenic Francisella, explaining phenotypic discrepancies in GSH utilization among Francisella spp. and revealing a critical role for GSH in the environmental niche of these bacteria.