Comparative Genomic Characterization of Francisella tularensis Strains Belonging to Low and High Virulence Subspecies

Abstract

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.

First indication for a functional CRISPR/Cas system in Francisella tularensis

“…We also found a first hint for a general tularensis, holarctica and mediasiatica seem to carry inactive CRISPR/Cas systems, exhibiting various gene deletions or pseudogenes. Similar findings were also published for the whole genome sequences of these strains (Champion et al, 2009). This process may be an adaptation of the strains to new habitats (e.g., like eukaryotic hosts) (Dobrindt et al, 2010;Ochman and Moran, 2001).…”

“…In Ft. mediasiatica, a chromsosmal rearrangement has occured in the respective region, probably caused by a recombination event of two ISFtu1 elements. The loss of genes may be a process of genomic pathoadaptation in response to changing or new habitats of the bacteria (Champion et al, 2009;Ochman and Moran, 2001;Svensson et al, 2005). The CRISPR/Cas system may be more relevant for the waterassociated species Ft. novicida and F. philomiragia, which gets in contact with a large number of different (invading) mobile genetic elements.…”

“…The genome sequences of strain U112, the clinical isolate 3523 (Australian strain) and Fx1 (Texas strain) are available, as well as some Ft. tularensis and F. philomiragia strains (Barabote et al, 2009;Beckstrom-Sternberg et al, 2007;Champion et al, 2009;Chaudhuri et 3 al., 2007;Larsson et al, 2005;Modise et al, 2012;Nalbantoglu et al, 2010;Petrosino et al, 2006;Rohmer et al, 2007;Siddaramappa et al, 2011;Sjodin et al, 2012;Zeytun et al, 2012). Ft. tularensis is a highly infectious zoonotic agent.…”

Phylogeographical pattern of Francisella tularensis in a nationwide outbreak of tularaemia in Norway, 2011

“…In addition to the 18 Norwegian and 29 selected Swedish strains, six common reference genomes were included (OR96246, FTNF002-00 [26], LVS, Tul7 [13], FSC200 [24], OSU18 [27], and the tree was rooted in FSC022 [28]. The phylogenetic tree was constructed in MEGA6 [29].…”

Francisella tularensis Molecular Typing Using Differential Insertion Sequence Amplification

“…Despite the subtype A.I and A.II strains having similar numbers of IS elements (6,20), there is considerable overall variation in chromosome architectures (18,23). The current study revealed that a 312-bp insertion or deletion derived from ISFtu1 was responsible for the size heterogeneity within the CR-derived amplicons in the subtype A.I and A.II strains but that discrimination between these two clades was still apparent.…”