Francisella tularensis is an extremely pathogenic bacterium

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Francisella tularensis is an extremely pathogenic bacterium that is capable of infecting a broad range of hosts (Ludu J. S., et al. 2008). Infection can occur via direct contact, ingestion or inhalation and is the causative agent of the zoonotic disease tularemia in humans (Robb CS, et al., 2010). The highly infectious nature of the bacteria is demonstrated by its ability to cause disease in a healthy human when exposed to as few as ten cells as well as the high mortality rate associated with infection via the pulmonary system (Robb CS, et al., 2010; Barker JR., 2009). Although recent molecular diagnostics research has enabled highly sensitive and specific diagnoses of infection, there remains no available vaccine for Francisella tularensis in North America (Tarnvik A. & Chu M.C., 2007; Barker J.R. et al., 2009). Immediate antibiotic treatment is moderately effective and available for some of the Francisella subspecies, yet there are many adverse effects of the treatment (Tarnvik A. & Chu M.C., 2007). The highly pathogenic nature of the organism along with its ability to avoid host cell immunity using an uncharacterized secretion system has lead recent researchers to examine Francisella in order to elicit further roles of secretion systems in bacterial virulence (Barker J.R. et al., 2009). F. tularensis subsp. novicida (Ftn) is used to study F. tularensis pathogenicity because of its low virulence in humans but high virulence in mice (Barker J. R. & Klose E. K., 2007). Additionally this subspecies has well established genetic techniques which facilitate ease of study.

1.2 Francisella tularensis virulence

The pathogenicity of a bacterial species is determined by its virulence factors, including general adhesion, colonization, antibacterial resistance, invasion, toxins or immune response repression mechanisms. Some previously described virulence factors in Francisella spp. are the tolC and tolC like homologues which act in a type I secretion system (T1SS) manner to provide bactericidal resistance (Gil H. et al., 2006). Similarly, Bina et al. (2008) showed that the AcrB RND efflux pump also contributed to Francisella spp. drug resistance (Bina et al., 2008). Together these papers, along with others, indicate an emerging importance in characterizing bacterial secretion systems given their widespread occurrence in pathogenic bacteria.

The high virulence of some Francisella strains is attributed to their ability to survive and replicate within human host macrophages. This replication of F. tularensis within host cells is dependent upon a secretion system that has been identified to be encoded by the genes of the Francisella pathogenicity island (FPI) (Barker JR., et al., 2009). F. tularensis uses the secretion system to escape from a macrophage phagosome into the cytosol, effectively evading the human immune system (Barker JR., et al., 2009). Once free, the bacteria are able to induce the caspase-1 dependent inflammasome leading to apoptosis and autophagosome engulfment (Barker, J.R. et al., 2009).

1.3 Francisella pathogenicity island (FPI)

The FPI encodes 16-19 genes with a 97% nucleotide identity across the Francisella subspecies and is found in duplicate within the highly virulent F. tularensis and F. holarctica species (Barker, J.R. 2009). Figure 1 shows a diagrammatic representation of the F. novicida FPI which encodes a number of gene products required for phagosome escape and cytosol replication.

Some of the FPI genes share homology with the type VI secretion system (T6SS) gene clusters in Vibrio cholera and Pseudomonas aeruginosa, however the levels of identity at the nucleotide sequence are low ( get a % identity for IglE). Two of the FPI proteins, VgrG and IglI are secreted into the cytosol of infected macrophages and have been shown to be required for phagosome escape, intramacrophage growth and inflammasome activation in gene knockout studies in mice (Barker, J.R. 2009). Mutagenesis studies of IglA, IglB, IglD and PdpA genes have shown that many of the FPI genes are required for intracellular growth and virulence of Francisella spp. while further studies of IglA and IglB have shown homology to T6SS proteins from Vibrio and Pseudomonas (Nano FE., et al., 2004; Nano FE., Schmerk C. 2007). Ludu J.S. et al. (2008) showed that the FPI protein PdpD was required for full virulence in F. tularensis Strain A however unlike most other FPI genes it was not needed in intramacrophage growth. Continued research on the genes encoded by the FPI is required to construct a broader understanding of the virulence mechanism of Francisella involving secretion system assisted macrophage escape. Although the essential role in virulence of many FPI genes has been examined, structural biology approaches have been limited, and the current model of the FPI secretion system remains uncharacterized.

Figure 1: Francisella novicida pathogenicity island. Diagrammatic representation of the F. novicida FPI with consensus nomenclature of FPI genes.

1.4 Secretion systems

Bacteria have evolved a collection of protein secretion systems that enable them to survive within hostile environments, invade and colonize eukaryotic cells or even engage in inter-bacterial warfare (Preston GM, 2007). To date, six unique gram-negative bacteria secretion systems have been identified to deliver proteins to both the extracellular environment or directly into host cytosol to modulate host cell growth and physiology (Record AR, 2011; Hayes CS, et al., 2010). Additionally there are other transport mechanisms available such as the post translational transfer of proteins via the SecA/SecY system, as well as the contrasting Tat system, which is able to translocate fully folded protein complexes with essential cofactors bound (Holland B, 2010). These mechanisms of protein translocation have been well characterized in three Nature papers, (28, 29, 30), and are adopted by some of the six secretion systems. Structural complexes of the ATPase SecA, protein translocation channel as well as additional information obtained from transitional state structures have driven the understanding of SecA/SecY and Tat secreting system (28, 29, 30).

Looking specifically to the first of the six secretion systems, the type I secretion system (T1SS) characterized in E. coli involves a relatively simple secretion mechanism transporting proteins to the external environment. T1SSs require three proteins, a cytoplasmic ABC ATPase (HlyB) complexed to a membrane anchored membrane fusion protein (MFP) (HlyD) which spans the periplasm of the gram-negative bacteria (Holland B, 2010). This system then recognizes the C-terminal signal sequence of haemolysin (HlyA) and secretes it through the recruited TolC outer membrane (OM) protein trimer (Holland B, 2010). As expected by the presence of the ABC ATPase, T1SSs require the hydrolysis of ATP for effective secretion (Koronakis, V., et al., 1991).

Type II secretion systems (T2SSs) are the best studied two-step mechanisms of secretion and are present in bacteria such as Vibrio cholera and Pseudomonas aeruginosa (Holland B, 2010). Two-step secretion systems involve a periplasmic intermediate which is transported across the cytoplasmic membrane of the bacteria via the Sec of Tat machines previously described (Holland B. 2010). Additional research by Francetic O, et al. (2007) shows that proteins can also be transported to the periplasm using the co-translational SRP mediated SecYEG translocon. The T2SSs involve an ATPase base, and multimeric OM translocon or secretin (Holland B, 2010). The overall structure is comprised of pilin like subunits which constitute the formation of the OM channel. An interesting observation of atypical T2SS genetic organization has been observed in P. aeruginosa (Wooldridge K. 2009). Atypical cases of T2SSs have the secretion system (SS) component genes spread throughout the chromosome and in some cases lack typical components of the T2SS complex (Wooldridge K. 2009). This variance in complete organization and conservation of SS machinery components suggests that a level of flexibility in the designation of specific bacterial secretion systems is observed.

Similar to type I and II, T5SSs T5SSs represent one of the largest groups of translocation systems in gram-negative bacteria and involve autotransporter polypeptides (ATs) and the Sec IM system (Holland B. 2010). Following Sec mediated IM transport, the C-terminal autotransporter domain spontaneously inserts into the OM via the formation of a β-barrel structure (Holland B. 2010). The AT domain then translocates the N-terminal domain (Holland B. 2010).

The TIII, TIV, TVI, secretion systems form surface complexes which deliver DNA and/or proteins to host cells via contact dependent mechanisms (Holland B. 2010). The structures for all of the type III secretion systems (T3SSs) components have been characterized to provide a injectisome structure model that is able to secrete various effectors through a flagella like channel (Hayes CS, 2010). Similar to flagella, the T3SS includes rings embedded within the IM and OM with a large entrance site at the cytosolic face of the IM (Hayes et al., 2010). Crucial gene products that resemble the molecular ruler protein FliK in flagella have been observed to control the extension of the injectisome needle length (Hayes et al., 2010). Gene knockout studies indicate continued needle extension and dissociation from the SS machinery without the FliK homologue YscP in Yersinia (Hayes et al., 2010). Research from the Galan laboratory described how an ATPase associated at the base of the T3SS recognize an N-terminal signal sequence of chaperone-effector complexes at the cytosolic entrance and unfold them prior to secretion (Akeda Y. et al., 2005; Hayes et al., 2010). Two key proteins are present at the needle tip and form a translocon pore in the host cell that is dependent on cell-cell contact to secrete the specific components of the translocon (Hayes et al., 2010).

T4SSs are involved in the secretion of protein, DNA or protein/DNA complexes via conjugal plasmid transfer in both a contact dependent and independent manner (Hayes et al., 2010). These SSs involve at least 12 characterized polypeptides including a terminal pilin-like extension through the periplasm to penetrate target cells (Hayes et al., 2010). A number of pathogens, such as Bordetella pertussi, use T4SSs to secrete toxic effector proteins into host prokaryotic cells and have been connected to the horizontal spread of antibiotic resistance (Hayes et al., 2010). The three main components of T4SSs are the cell surface adhesion or pili like cell contact mediators, the secretion channel within the donor cell envelope and a Type IV coupling protein (T4CP) that recognizes substrates at the cytosolic base of the channel (Hayes et al., 2010). Although the channel assembly and structure are not completely characterized, the T4SS from Agrobacterium tumefaciens has provided a general model involving 3 ATPases at the base of the cage like component spanning the entire envelope from IM to OM.

As illustrated by the current understanding of the first five secretion systems, combining structural and molecular characterization of core components has been critical in constructing secretion system models as a first step for guiding future research. The recently discovered type VI secretion system (TSS) remains poorly characterized, however subsequent structural characterization of the core components will likely provide key information on the mechanism and importance of T6SSs in bacterial virulence and survival.

1.5 Type VI secretion systems

Type VI secretion systems (T6SSs) have recently been discovered as important virulence factors in organisms such as Cholera, Yersinia and Pseudomonas (Holland B. 2010). Initial examination of core T6SSs components revealed similarities to components found in the DNA delivery tail of bacteriophages P22 and T4 suggesting an evolutionary relationship (Hayes et al. 2010, Leiman PG. et al., 2009). The conserved VgrG gene in T6SSs contains a similar N-terminal and central region to the two components of the bacteriphage tailspike device (Hayes et al., 2010). Additional homology exists between the baseplate proteins of the two systems as well as between the T6SS hexameric Hcp protein and the bacteriophage tail tube proteins (Hayes et al., 2010). A general T6SS model is adopted from Kanehisa Laboratories (2010) and shows core components with putative interactions in figure 2.

Figure 2. Current model of type VI secretion system. Adapted from Kanehisa Laboratories (2010) showing the core components of T6SSs with a proposed association across bacterial and host membranes. (need to redo and label OM IM ect)

Many T6S clusters are found within pathogenicity islands such as the Hcp Secretion island (HSI) of Pseudomonas aeruginosa or pheU of enteroaggregative Escherichia coli which act as model T6SSs (Cascales E. 2008). Three T6SS gene clusters have been identified in P. aeruginosa which deliver a toxic effector protein, Tse2, into other bacteria (Hood, et al.). The core components of T6SSs are a conserved hexameric AAA+ ATPase, ClpV, the two VgrG and Hcp secreted effectors, IcmF and IcmH (DotU) T4SS-like components and an outer membrane lipoprotein (Hayes et al., 2010).

The focus of this thesis is on one of the blah blah blah SciN OM lipoprotein from enteroaggregative E. coli was described as an essential T6SS component required for biofilm formation and Hcp-like SciD protein secretion (Aschtgen MS, et al., 2008). SciN was determined to be localized in the OM exposed to the periplasm via selective detergent solubilisation and isopycnic sucrose sedimentation gradients (Aschtgen MS. et al., 2008). Gene cluster analysis of hypothetical lipoproteins in other bacteria known to contain T6SSs lead to the identification of a SciN orthologue in P. aeruginosa, PA0080. The FPI also encodes an OM lipoprotein, IglE, which may be a functional analogue of the T6SS component TssJ.

1.6 Francisella IglE

IglE is a putative lipoprotein (14 469.5 Da; 125 amino acids) required for secretion and intramacrophage growth (Robb C. et al., 2010). Although little to no sequence identity exists between IglE and other T6SS genes, IglE does share orthology with the previously described lipoprotein family of T6SSs in enteroaggregative E.coli and P. aeruginosa (Kanehisa M, et al., 2004). Many of the conserved core components of T6SSs have been identified within the FPI, such as VgrG and Hcp like secreted proteins and further examination of the structural biology of the putative lipoprotein could provide insight into the machinery assembly of the FPI as well as further the characterization of T6SSs.

Robb C. et al. (2010) solved the crystal structure of IglE which contained two helices between the second and third β-strands, as seen in Figure 3. Examination of the structure suggested that this protein may exist as a dimer in solution. A small putative dimerisation interface was identified. Small-angle X-ray scattering (SAXS) was used to observe the IglE dimer in solution (Robb C. unpublished). Looking back to the model for T6SS in Figure 2, the observed dimerization of the IglE lipoprotein could provide a critical functional role in anchoring the FPI secretion system into the bacterial OM. Mutagenesis of the hydrophobic residues thought to be involved in dimerisation was performed to examine structural effects of disrupting the dimer in vitro. A member of the conserved T6SS components TssJ was also structurally characterised to demonstrate structural identity with IglE

Figure 3: Native IglE structure and dimer interface. Key hydrophobic residues targeted by mutagenesis to disrupt dimerization. Close up interaction of the IYV residues between the two domains.