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Francisella tularensis is a highly virulent, intracellular pathogen that causes the disease tularaemia. A research surrogate for F. tularensis is Francisella novicida, which causes a tularaemia-like disease in mice, grows similarly in macrophages, and yet is unable to cause disease in humans. Both Francisella species contain a cluster of genes referred to as the Francisella pathogenicity island (FPI). Pathogenicity determinant protein A (PdpA), encoded by the pdpA gene, is located within the FPI and has been associated with the virulence of Francisella species. In this work we examined the properties of PdpA protein expression and localization as well as the phenotype of a F. novicida pdpA deletion mutant. Monoclonal antibody detection of PdpA showed that it is a soluble protein that is upregulated in iron-limiting conditions and undetectable in an mglA or mglB mutant background. Deletion of pdpA resulted in a strain that was highly attenuated for virulence in chicken embryos and mice.

Several genes contained in the Francisella pathogenicity island (FPI) encode proteins needed for intracellular growth and virulence of Francisella tularensis. The pdpA gene is the first cistron in the larger of the two operons found in the FPI. In this work we studied the intracellular growth phenotype of a Francisella novicida mutant in the pdpA gene. The [DELTA]pdpA strain was capable of a small amount of intracellular replication but, unlike wild-type F. novicida, remained associated with the lysosomal marker LAMP-l, suggesting that PdpA is necessary for progression from the early phagosome phase of infection. Strains with in cis complementation of the [DELTA]pdpA lesion showed a restoration of intracellular growth to wild-type levels. Infection of macrophages with the [DELTA]pdpA mutant generated a host-cell mRNA profile distinct from that generated by infection with wild-type F. novicida. The transcriptional response of the host macrophage indicates that PdpA functions directly or indirectly to suppress macrophage ability to signal via growth factors, cytokines and adhesion ligands.

Francisella tularensis is a highly infectious, facultative intracellular bacterial pathogen that is the causative agent of tularemia. Nearly a century ago, researchers observed that tularemia was often fatal in North America but almost never fatal in Europe and Asia. The chromosomes of F. tularensis strains carry two identical copies of the Francisella pathogenicity island (FPI), and the FPIs of North America-specific biotypes contain two genes, anmK and pdpD, that are not found in biotypes that are distributed over the entire Northern Hemisphere. In this work, we studied the contribution of anmK and pdpD to virulence by using F. novicida, which is very closely related to F. tularensis but which carries only one copy of the FPI. We showed that anmK and pdpD are necessary for full virulence but not for intracellular growth. This is in sharp contrast to most other FPI genes that have been studied to date, which are required for intracellular growth. We also showed that PdpD is localized to the outer membrane. Further, overexpression of PdpD affects the cellular distribution of FPI-encoded proteins IglA, IglB, and IglC. Finally, deletions of FPI genes encoding proteins that are homologues of known components of type VI secretion systems abolished the altered distribution of IglC and the outer membrane localization of PdpD.

Hopanoids are bacterial surrogates of eukaryotic membrane sterols and among earth's most abundant natural products. Their molecular fossils remain in sediments spanning more than a billion years. However, hopanoid metabolism and function are not fully understood. B urkholderia species are environmental opportunistic pathogens that produce hopanoids and also occupy diverse ecological niches. We investigated hopanoids biosynthesis in B urkholderia cenocepacia by deletion mutagenesis and structural characterization of the hopanoids produced by the mutants. The enzymes encoded by hpnH and hpnG were essential for production of all C35 extended hopanoids, including bacteriohopanetetrol ( BHT), BHT glucosamine and BHT cyclitol ether. Deletion of hpnI resulted in BHT production, while Δ hpnJ produced only BHT glucosamine. Thus, HpnI is required for BHT glucosamine production while HpnJ is responsible for its conversion to the cyclitol ether. The Δ hpnH and Δ hpnG mutants could not grow under any stress condition tested, whereas Δ hpnI, Δ hpnJ and Δ hpnK displayed wild-type growth rates when exposed to detergent, but varying levels of sensitivity to low pH and polymyxin B. This study not only elucidates the biosynthetic pathway of hopanoids in B . cenocepacia, but also uncovers a biosynthetic role for the conserved proteins HpnI, HpnJ and HpnK in other hopanoid-producing bacteria. [ABSTRACT FROM AUTHOR]