SUMMARY Francisella tularensis is a facultative intracellular gram-negative pathogen and the etiological agent of the zoonotic disease tularemia. Recent advances in the field of Francisella genetics have led to a rapid increase in both the generation and subsequent characterization of mutant strains exhibiting altered growth and/or virulence characteristics within various model systems of infection. In this review, we summarize the major properties of several Francisella species, including F. tularensis and F. novicida, and provide an up-to-date synopsis of the genes necessary for pathogenesis by these organisms and the determinants that are currently being targeted for vaccine development.
Background Francisella tularensis is a Gram-negative coccobacillus that causes the febrile illness tularemia. Subspecies that are pathogenic for humans include those comprising the type A (subspecies tularensis) or type B (subspecies holarctica) biovars. An attenuated live vaccine strain (LVS) developed from a type B isolate has previously been used to vaccinate at-risk individuals, but offers limited protection against high dose (>1000 CFUs) challenge with type A strains delivered by the respiratory route. Due to differences between type A and type B F. tularensis strains at the genetic level, it has been speculated that utilization of an attenuated type A strain as a live vaccine might offer better protection against homologous respiratory challenge compared with LVS. Here, we report the construction and characterization of an unmarked ΔpurMCD mutant in the highly virulent type A strain Schu S4.Methodology/Principal FindingsGrowth of Schu S4 ΔpurMCD was severely attenuated in primary human peripheral blood monocyte-derived macrophages and in the A549 human lung epithelial cell line. The Schu S4 ΔpurMCD mutant was also highly attenuated in mice when delivered via either the intranasal or intradermal infection route. Mice vaccinated intranasally with Schu S4 ΔpurMCD were well protected against high dose intradermal challenge with virulent type A or type B strains of F. tularensis. However, intranasal vaccination with Schu S4 ΔpurMCD induced tissue damage in the lungs, and conferred only limited protection against high dose Schu S4 challenge delivered by the same route. The level of protection observed was similar to that conferred following vaccination with wild-type LVS or the analogous LVS ΔpurMCD mutant.Conclusions/SignificanceCollectively, these results argue that development of the next generation live attenuated vaccine for Francisella should be based on use of the less pathogenic type B biovar rather than the more reactogenic type A biovar.
Lung surfactant protein D (SP-D) binds toPulmonary surfactant proteins A and D (SP-A and SP-D, respectively) participate in the lung innate and adaptive immune responses against multiple pathogens through opsonization and agglutination of microorganisms, blockade of receptor-ligand interactions, and/or direct effects on host cells, particularly macrophages (5,27). Experiments using mice with homozygous null alleles for the SP-A or SP-D genes provide direct evidence in support of these phenomena (11,13,(17)(18)(19)(20).Both SP-A and SP-D have been implicated in the pathogenesis of tuberculosis. Our work has demonstrated that SP-A increases the phagocytosis of Mycobacterium tuberculosis by human macrophages by up-regulating the macrophage mannose receptor (MR) (2, 10) and reduces NADPH oxidase activity in these cells (6). In contrast, SP-D, through its carbohydrate recognition domain (CRD), binds to the terminal mannose caps of the M. tuberculosis surface lipoglycan lipoarabinomannan (ManLAM) and causes agglutination of the bacilli and inhibition of bacterial uptake by macrophages (7). However, the reduced uptake was not dependent on bacterial agglutination (8). The presence of SP-D also inhibited the intracellular growth of M. tuberculosis in macrophages (8). M. tuberculosis evades several host defense mechanisms including by inhibiting fusion of its own phagosome with lysosomes in macrophages (1) and avoiding the potential stimulation of intracellular oxidant production during phagocytosis (26).We hypothesized that SP-D inhibits the intracellular growth of M. tuberculosis in macrophages by altering intracellular events that restrict mycobacterial growth following phagocytosis. In this study, we examined whether SP-D coating of M. tuberculosis modulates two major macrophage microbicidal mechanisms: generation of a respiratory burst and regulation of phagosome-lysosome (P-L) fusion events.Recombinant rat SP-D (referred to as SP-D throughout the paper), produced in CHO cells and purified on a mannoseSepharose matrix (22, 23), was used in the different assays at a concentration of 0.5 (nonagglutinating for M. tuberculosis) and/or 5.0 (agglutinating for M. tuberculosis) g/ml. Since SP-D had been shown to inhibit the intracellular growth of M. tuberculosis in macrophages, we first determined whether SP-D had a direct microbicidal effect on M. tuberculosis by performing CFU assays following incubation of the bacilli in the absence or presence of SP-D. No significant effect of SP-D on the viability of bacteria at either 0.5 or 5.0 g/ml was observed (n ϭ 2; data not shown).SP-D does not induce a respiratory burst during phagocytosis of M. tuberculosis in human macrophages. We next determined whether phagocytosis of M. tuberculosis by human macrophages in the presence of SP-D would stimulate the production of reactive oxygen intermediates by using the 2Ј,7Ј-dichlorofluorescein (DCF) assay (6). Monocyte-derived macrophages (MDMs) were prepared from healthy human volunteers as described previously (24). MDM monolayers on ti...
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