Francisella tularensis Has a Significant Extracellular Phase in Infected Mice

Francisella tularensis, which causes the disease tularemia, has been classified by the Centers for Disease Control and Prevention as a category A agent, 1 of 6 that would most seriously affect public health if they were used as a biological weapon [1]. Several subspecies of this highly infectious gram-negative bacterium exist, 2 of which are relevant human pathogens. F. tularensis subspecies tularensis (also known as type A) displays the highest virulence in humans, whereas F. tularensis subspecies holarctica (type B) causes a milder form of tularemia. An attenuated live vaccine strain (LVS) of F. tularensis has been generated from a type B isolate. This strain is avirulent in humans but retains the ability to cause lethal disease in mice, making it a useful model for studying the pathogenesis of tularemia. The prototypical laboratory strain of type A F. tularensis, Schu S4, is fully virulent in both mice and humans [2].

F. tularensis is known for its ability to enter and replicate within host cells [3]. Growth of Francisella within macrophages may be essential for virulence, because F. tularensis organisms lacking a protein highly expressed during intramacrophage growth cannot replicate to high numbers within macrophages in vitro and fail to cause lethal disease in mice. Shortly after entry into the macrophage, F. tularensis evades the normal killing mechanisms, escaping from the phagosome into the cytoplasm, where it replicates freely [2]. Thus, Francisella exploits the host macrophage, utilizing it as a protective niche wherein it can proliferate. Based on these observations, a long-standing belief in the field of Francisella research is that when in the bloodstream of infected hosts, the bulk of the bacteria reside intracellularly. Indeed, a previous study suggested that ∼99% of F. tularensis organisms in the blood of bacteremic mice are cell associated [4]. Here, we report findings that strongly refute this conclusion. Instead, we noted that F. tularensis-infected mice presenting with bacteremia contain substantial numbers of viable organisms in the plasma fraction of their blood.

Materials and methods. F. tularensis LVS (ATCC 29684) and strain Schu S4 (US Army Medical Research Institute for Infectious Diseases) were cultured as described elsewhere [5].

All murine studies were approved by the Institutional Animal Care and Use Committees of Stony Brook University and Albany Medical College. Six-to 8-week-old mice (Taconic Laboratories) were infected intradermally or intranasally with either the LVS or Schu S4 strain. In one experiment, mice were inoculated intradermally with 10⁷ cfu of F. tularensis LVS made to express green fluorescent protein (GFP) by transformation with pFNLTP6-gro-gfp, a plasmid provided by T. Zahrt (Medical College of Wisconsin).

Blood from killed mice was collected by cardiac puncture or retroorbital bleeding using sodium citrate as an anticoagulant. The plasma and cellular portions were separated by centrifugation at 200 g for 5 min. The plasma was removed to a separate tube, and the cells were washed twice in PBS. The supernatant of each wash was added to the plasma, and the cellular pellet was lysed in 1 mL of water. No host cells were present in the plasma fraction as determined by microscopy. Dilutions of the cellular and plasma fractions were plated on Mueller-Hinton II blood agar to enumerate the numbers of F. tularensis in each. Omission of the washing steps had little effect on results, indicating that bacteria were not liberated from cells during this procedure.

To determine whether F. tularensis could grow in murine or human blood ex vivo, 10⁴ cfu of the LVS or the Schu S4 strain was inoculated into heparinized whole blood from either species. The blood andF. tularensis were cultured together at 37°C with shaking at 100 rpm for 24 h and processed as described above. No human donors had F. tularensis-specific antibodies, as assessed by Western blot analysis.

LVS organisms expressing GFP in murine blood were viewed by epifluorescence and phase contrast microscopy with a Zeiss Axioplan2 microscope. Images were captured using a Spot camera (Diagnostic Instruments) and processed using Adobe Photoshop (version 6.0).

Results. When C3H/HeN mice were infected intradermally with a lethal dose of 10⁷ cfu of the LVS, F. tularensis organisms were detected in their blood as early as 1 day after inoculation. Although the number of colony-forming units per milliliter of whole blood varied from mouse to mouse, substantial numbers of F. tularensis were found in the blood of all mice up to 5 days after the initial infection (table 1). No mice survived for longer than 5 days at this infectious dose. Sublethal intradermal inocula of the LVS consisting of 10⁵ (data not shown) and 10³ cfu resulted in sustained bacteremia as well, beginning 2 days after inoculation and ranging from 5.4 × 10² to 1.9 × 10⁵ cfu/mL. However, the persistence of bacteria in the blood decreased with the infectious dose, with no bacteria detectable 4 days after inoculation with 10³ cfu (table 1).

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Table 1.

Bacteremia in C3H/HeN mice infected with Francisella tularensis live vaccine strain (LVS) or Schu S4.

To establish whether route of inoculation affects the duration and level of bacteremia, mice were inoculated intranasally with highly lethal (10⁶ cfu) as well as sublethal (10³ and 10² cfu) doses of F. tularensis LVS. Mice receiving a lethal inoculum of the LVS presented with bacteremia from days 2 to 7 after infection, with no mice surviving longer than 7 days. F. tularensis LVS was not detected in the blood of sublethally infected mice until day 3 (10³ cfu) or day 4 (10² cfu) and persisted until at least day 7 (table 1 and data not shown). Intranasal inoculation with a lethal dose (10² cfu) of fully virulent F. tularensis strain Schu S4 yielded similar results; bacteria were not found in the blood until 4 days after inoculation and persisted until the sixth day after inoculation, after which no mice survived (table 1).

To determine whether almost all F. tularensis organisms in the blood of infected mice are cell associated, as has been suggested [4], we separated the cellular and plasma fractions of whole blood taken from the tularemic mice discussed above. In total, F. tularensis was detected in the blood of 48 C3H/HeN mice. In all of these instances, extracellular bacteria were detected, and they constituted the majority of the bacteria recovered in 42 of the 48 cases. In the bacteremic mice, the average percentage of extracellular F. tularensis was 75%, ranging from 31% to 100%. This distribution was observed in mice inoculated intradermally or intranasally, with sublethal or lethal doses, and with attenuated or fully virulent F. tularensis (table 1). This observation is not specific to C3H/HeN mice, because BALB/c and C57BL/6 mice infected intradermally with lethal or sublethal doses of the LVS also presented with sustained bacteremia characterized by an abundance of extracellular organisms in the blood (data not shown). We visualized this phenomenon using LVS organisms engineered to express GFP. Examination by fluorescence microscopy of whole blood drawn from mice infected with a lethal dose of this strain clearly showed both cell-and plasma-associated F. tularensis (figure 1).

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Figure 1.

Observation of Francisella tularensis both intra-and extracellularly in the blood of tularemic mice. Whole blood was collected from C3H/HeN mice that had been infected with 10⁷ green fluorescent protein- expressing F. tularensis live vaccine strain (LVS) organisms for 2 days, slides were prepared, and bacteria were visualized by fluorescence and phase-contrast microscopy. Panels represent the epifluorescent image alone (C) or the epifluorescent image superimposed on a phase contrast microscopic image of the same field (A, B, and D). A, Extracellular F. tularensis LVS, indicated by arrows. B, LVS organisms associated with a blood leukocyte. C and D, Low-magnification view of F. tularensis LVS organisms in the blood, none of which are within leukocytes.

Over a 24-h period, both the LVS and the Schu S4 strain grew in murine whole blood ex vivo, increasing an average of 22-fold and 30-fold, respectively, in 5 separate experiments (figure 2A). Under comparable conditions using human whole blood, only F. tularensis Schu S4 showed a modest ability to replicate. In 5 experiments, Schu S4 bacteria increased an average of 4-fold (figure 2B). As was noted in vivo, the majority of bacteria recovered from the blood of either species were plasma associated (figure 2). LVS organisms did not replicate in human blood reconstituted with autologous heat-inactivated plasma, indicating that their failure to grow was not due to complement-mediated killing.

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Figure 2.

Growth of Francisella tularensis in human and murine whole blood. In each experiment, the live vaccine strain (LVS;) or Schu S4 strain (D)of F. tularensis was inoculated into an aliquot of the same sample of murine (A) or human (B) whole blood. For each host species, results of 5 experiments using different donors are shown. After 24 h, the cellular and plasma portions of the blood were separated and plated to enumerate F. tularensis organisms in each. Growth of the Schu S4 strain in human blood was significantly greater than that of the LVS by a paired t test (P=.0035). The growth of the 2 strains in murine blood was not significantly different. Note the difference in the Y-axis scale between the 2 panels. Nos. shown in parentheses next to each data point represent the percentage of F. tularensis organisms recovered that were plasma associated. Dotted lines represent the approximate starting amount of F. tularensis organisms inoculated (10⁴ cfu).

Discussion. Our observations refute a long-standing paradigm within the tularemia field, namely, that F. tularensis in the blood of infected hosts is primarily cell associated. The ability of F. tularensis to replicate within monocytes and macrophages, together with a previous publication that suggests that ∼99% of all F. tularensis organisms in the blood of tularemic mice are cell associated [4], have engendered this belief.

Recently, a study of human volunteers who were vaccinated with the LVS found that the bacterium was not detected in the blood of any subjects throughout a 35-day postvaccination time course [6]. This finding, coupled with the fact that wild-type Francisella strains have been recovered from the blood of humans [7], suggests that the capacity of virulent strains to survive in the blood may be a major reason for their ability to cause human disease. Mice infected with both the LVS and Schu S4 strain of F. tularensis presented with sustained bacteremia, the degree and duration of which generally increased with greater infectious doses. When the LVS and Schu S4 strain were administered to mice intranasally at comparable doses, the Schu S4 strain reached much higher numbers in the blood (table 1), a property that perhaps contributes to its greater virulence. Together, these data support the notion that survival and replication of F. tularensis in the blood of infected hosts is important to the development of disease.

That F. tularensis has a marked extracellular phase in the infected host adds to our understanding of the pathogenesis of tularemia. Despite possessing an atypical and relatively inert lipopolysaccharide [2], F. tularensis is capable of eliciting a host inflammatory response. In vivo studies have implicated tumor necrosis factor-α, interferon-γ, and interleukin-12 as important mediators of the murine response to infection with Francisella [8, 9]. Although relatively little is known about the interactions between human cells and Francisella, human peripheral blood monocytes [10] and primary endothelial cells [5] respond to live or killed preparations of F. tularensis LVS by producing a selected array of proinflammatory chemokines and cytokines. The concept that F. tularensis organisms in the blood of infected hosts reside almost entirely within leukocytes makes it difficult to envision a scenario in which cells other than the infected leukocytes themselves could come into contact with the bacteria. However, our findings clearly support a situation in which F. tularensis could interact with a wide spectrum of cells in the bloodstream, initiating a pronounced host inflammatory response in vivo. Furthermore, our results are important in the context of the host antibody response to Francisella. Humoral immunity has been implicated as a factor in protection of mice against type B strains of F. tularensis [11- 13], and it is likely that recognition of the organisms by antibody would occur during their extracellular phase.

Both F. tularensis LVS and Schu S4 grew well in murine whole blood ex vivo but not in murine plasma (data not shown), suggesting that they require host cells or cellular components to replicate in blood. The LVS failed to increase in numbers in human blood ex vivo, although cultured human monocytes and macrophages support its replication [10]. Perhaps, then, F. tularensis LVS is susceptible to as-yet-undefined killing mechanisms in human blood, preventing it from growth. The fact that no LVS organisms were found in the blood of vaccinated individuals supports this notion [6]. Unexpectedly, the type A Schu S4 strain grew more poorly in human blood than in murine blood, although it causes disease in both hosts. The reason for this difference is unknown, but our data rule out the possibility that it is mediated by complement or specific antibodies. Based on our findings, we feel that F. tularensis in the blood of infected hosts likely is taken up by and replicates within leukocytes, eventually escaping into the plasma where it propagates a cycle of infection, escape, and reinfection.

The fact that differences in the capacity of F. tularensis to grow in murine and human blood ex vivo do exist suggests that caution should be exercised before conclusions about human tularemia are drawn from findings in mice. Nonetheless, the observation that there are substantial numbers of extracellular F. tularensis within murine hosts should be considered when designing anti- Francisella therapies and vaccines.

• Received October 11, 2006.
• Accepted February 2, 2007.

• © 2007 by the Infectious Diseases Society of America