The Francisella pathogenicity island (FPI) encodes proteins thought to compose a type VI secretion system (T6SS) that is required for the intracellular growth of Francisella novicida. In this work we used deletion mutagenesis and genetic complementation to determine that the intracellular growth of F. novicida was dependent on 14 of the 18 genes in the FPI. The products of the iglABCD operon were localized by the biochemical fractionation of F. novicida, and Francisella tularensis LVS. Sucrose gradient separation of water-insoluble material showed that the FPI-encoded proteins IglA, IglB and IglC were found in multiple fractions, especially in a fraction that did not correspond to a known membrane fraction. We interpreted these data to suggest that IglA, IglB and IglC are part of a macromolecular structure. Analysis of published structural data suggested that IglC is an analogue of Hcp, which is thought to form long nanotubes. Thus the fractionation properties of IglA, IglB and IglC are consistent with the current model of the T6SS apparatus, which supposes that IglA and IglB homologues form an outer tube structure that surrounds an inner tube composed of Hcp (IglC) subunits. Fractionation of F. novicida expressing FLAG-tagged DotU (IcmH homologue) and PdpB (IcmF homologue) showed that these proteins localize to the inner membrane. Deletion of dotU led to the cleavage of PdpB, suggesting an interaction of these two proteins that is consistent with results obtained with other T6SSs. Our results may provide a mechanistic basis for many of the studies that have examined the virulence properties of Francisella mutants in FPI genes, namely that the observed phenotypes of the mutants are the result of the disruption of the FPI-encoded T6SS structure.

Tularemia is a geographically widespread, severely debilitating, and occasionally lethal disease in humans. It is caused by infection by a gram-negative bacterium, Francisella tularensis. In order to better understand its potency as an etiological agent as well as its potential as a biological weapon, we have completed draft assemblies and report the first complete genomic characterization of five strains belonging to the following different Francisella subspecies (subsp.): the F. tularensis subsp. tularensis FSC033, F. tularensis subsp. holarctica FSC257 and FSC022, and F. tularensis subsp. novicida GA99-3548 and GA99-3549 strains. Here, we report the sequencing of these strains and comparative genomic analysis with recently available public Francisella sequences, including the rare F. tularensis subsp. mediasiatica FSC147 strain isolate from the Central Asian Region. We report evidence for the occurrence of large-scale rearrangement events in strains of the holarctica subspecies, supporting previous proposals that further phylogenetic subdivisions of the Type B clade are likely. We also find a significant enrichment of disrupted or absent ORFs proximal to predicted breakpoints in the FSC022 strain, including a genetic component of the Type I restriction-modification defense system. Many of the pseudogenes identified are also disrupted in the closely related rarely human pathogenic F. tularensis subsp. mediasiatica FSC147 strain, including modulator of drug activity B (mdaB) (FTT0961), which encodes a known NADPH quinone reductase involved in oxidative stress resistance. We have also identified genes exhibiting sequence similarity to effectors of the Type III (T3SS) and components of the Type IV secretion systems (T4SS). One of the genes, msrA2 (FTT1797c), is disrupted in F. tularensis subsp. mediasiatica and has recently been shown to mediate bacterial pathogen survival in host organisms. Our findings suggest that in addition to the duplication of the Francisella Pathogenicity Island, and acquisition of individual loci, adaptation by gene loss in the more recently emerged tularensis, holarctica, and mediasiatica subspecies occurred and was distinct from evolutionary events that differentiated these subspecies, and the novicida subspecies, from a common ancestor. Our findings are applicable to future studies focused on variations in Francisella subspecies pathogenesis, and of broader interest to studies of genomic pathoadaptation in bacteria.

ABSTRACTPrior to the introduction ofHaemophilus influenzaetype b (Hib) conjugate vaccines, invasive Hib disease affected almost exclusively children. According to some recent studies, in the postvaccine era, adults, the elderly, and immunocompromised persons can be affected more often than children. As the production of type-specific anti-capsular polysaccharide antibodies is the major defense mechanism against Hib, individuals with defects in humoral immune responses have high susceptibility to infections caused by Hib. We hypothesized that nonvaccinated adults with chronic conditions causing immunosuppression may lack protective antibody to Hib. We assessed serum anti-Hib IgG levels and bactericidal activity in 59 patients with chronic renal failure, 30 patients with type 2 diabetes mellitus, 28 patients with chronic obstructive pulmonary disease (COPD), and 20 patients with multiple myeloma compared to 32 healthy controls of similar age. Considering antibody at >0.15 μg/ml as the protective correlate in unvaccinated individuals, we detected subprotective Hib antibody levels in 29% of chronic renal failure, 20% of diabetes, 14% of COPD, and 55% of myeloma patients compared to 3% of healthy controls. Additionally, 70% of myeloma and 58% of chronic renal failure patients did not have detectable serum bactericidal activity against Hib. Among individuals with severe diseases causing secondary immunodeficiency, patients with multiple myeloma and chronic renal failure are at an increased risk of invasive Hib disease. Considering that Hib continues to circulate in the population, this study provides a rationale for the immunization of some adult patients with secondary immunodeficiency with the pediatric Hib vaccine to achieve protective immunity.

We examined the utility of infecting chicken embryos as a means of evaluating the virulence of different Francisella sp. strains and mutants. Infection of 7-day-old chicken embryos with a low dose of F. novicida or F. tularensis subsp. holarctica live vaccine strain (LVS) resulted in sustained growth for 6 days. Different doses of these two organisms were used to inoculate chicken embryos to determine the time to death. These experiments showed that wild-type F. novicida was at least 10,000-fold more virulent than the LVS strain. We also examined the virulence of several attenuated mutants of F. novicida, and they were found to have a wide range of virulence in chicken embryos. Fluorescent microscopic examination of infected chicken embryo organs revealed that F. tularensis grew in scattered foci of infections, and in all cases the F. tularensis appeared to be growing intracellularly. These results demonstrate that infection of 7-day-old chicken embryos can be used to evaluate the virulence of attenuated F. tularensis strains.

Francisella novicida is a gram-negative pathogen that can induce disease in mice that mimics human tularemia, and is nearly identical to Francisella tularensis at the genomic level. In this work a number of antibiotic marker cassettes that incorporate a strong F. novicida promoter is constructed, which greatly enhances selection in F. novicida and F. tularensis. Two low-copy plasmid vectors based on a broad-host-range plasmid, and an integrating vector have also been made, and these can be used for genetic complementation. Two general approaches to deletion mutagenesis in F. novicida is also described.

High prevalence of invasive Hia disease among North American Aboriginal populations is more likely related to exposure than to inadequate immunity.

ABSTRACTAdult chronic renal failure patients undergoing hemodialysis are at an increased risk of invasiveHaemophilus influenzaetype b (Hib) disease due to the lack of functionally active anti-Hib antibodies. The pediatric Hib polysaccharide-protein conjugate vaccine is highly immunogenic in these patients and can provide protection against invasive Hib infection for at least 1 year.

The highly infectious bacteria, Francisella tularensis, colonize a variety of organs and replicate within both phagocytic as well as non-phagocytic cells, to cause the disease tularemia. These microbes contain a conserved cluster of important virulence genes referred to as the Francisella Pathogenicity Island (FPI). Two of the most characterized FPI genes, iglC and pdpA, play a central role in bacterial survival and proliferation within phagocytes, but do not influence bacterial internalization. Yet, their involvement in non-phagocytic epithelial cell infections remains unexplored. To examine the functions of IglC and PdpA on bacterial invasion and replication during epithelial cell infections, we infected liver and lung epithelial cells with F. novicida and F. tularensis ‘Type B’ Live Vaccine Strain (LVS) deletion mutants (ΔiglC and ΔpdpA) as well as their respective gene complements. We found that deletion of either gene significantly reduced their ability to invade and replicate in epithelial cells. Gene complementation of iglC and pdpA partially rescued bacterial invasion and intracellular growth. Additionally, substantial LAMP1-association with both deletion mutants was observed up to 12 h suggesting that the absence of IglC and PdpA caused deficiencies in their ability to dissociate from LAMP1-positive Francisella Containing Vacuoles (FCVs). This work provides the first evidence that IglC and PdpA are important pathogenic factors for invasion and intracellular growth of Francisella in epithelial cells, and further highlights the discrete mechanisms involved in Francisella infections between phagocytic and non-phagocytic cells.