Phenotypic and genomic analysis of hypervirulent human-associated Bordetella bronchiseptica - PubMed (original) (raw)

Phenotypic and genomic analysis of hypervirulent human-associated Bordetella bronchiseptica

Umesh Ahuja et al. BMC Microbiol. 2012.

Abstract

Background: B. bronchiseptica infections are usually associated with wild or domesticated animals, but infrequently with humans. A recent phylogenetic analysis distinguished two distinct B. bronchiseptica subpopulations, designated complexes I and IV. Complex IV isolates appear to have a bias for infecting humans; however, little is known regarding their epidemiology, virulence properties, or comparative genomics.

Results: Here we report a characterization of the virulence of human-associated complex IV B. bronchiseptica strains. In in vitro cytotoxicity assays, complex IV strains showed increased cytotoxicity in comparison to a panel of complex I strains. Some complex IV isolates were remarkably cytotoxic, resulting in LDH release levels in A549 cells that were 10- to 20-fold greater than complex I strains. In vivo, a subset of complex IV strains was found to be hypervirulent, with an increased ability to cause lethal pulmonary infections in mice. Hypercytotoxicity in vitro and hypervirulence in vivo were both dependent on the activity of the bsc T3SS and the BteA effector. To clarify differences between lineages, representative complex IV isolates were sequenced and their genomes were compared to complex I isolates. Although our analysis showed there were no genomic sequences that can be considered unique to complex IV strains, there were several loci that were predominantly found in complex IV isolates.

Conclusion: Our observations reveal a T3SS-dependent hypervirulence phenotype in human-associated complex IV isolates, highlighting the need for further studies on the epidemiology and evolutionary dynamics of this B. bronchiseptica lineage.

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Figures

Figure 1

Figure 1

Cytotoxicity of complex I and complex IV B. bronchiseptica isolates. A. HeLa, B. J774A.1, or C. A549 cells were infected with the indicated strains at a multiplicity of infection (MOI) of 50 in 24-well plates for 3 h. Following infection, release of lactate dehydrogenase (LDH) into culture medium was measured as described in Materials and Methods. Complex I and complex IV strains are designated by blue or red bars, respectively. P values were calculated by an unpaired two-tailed Student's t test.

Figure 2

Figure 2

Time course cytotoxicity assays. A. HeLa, B. J774A.1, or C. A549 cells were infected with the indicated strains at a multiplicity of infection (MOI) of 50 in 12-well plates. Aliquots of culture supernatants were removed at the indicated times and lactate dehydrogenase (LDH) levels were measured as described in Materials and Methods. Complex I and complex IV strains are designated by blue or red lines, respectively. Due to repeated sampling of culture medium for LDH release assays, we consistently observe a slight increase in cytotoxicity measured in kinetic experiments vs. single time point assays as shown in Figure 1. The differences range from none to less than 20 %, depending on the cytotoxicity of the isolate. Error bars represent standard errors for measurements from at least three independent experiments.

Figure 3

Figure 3

Roles of the bsc T3SS and the BteA effector in cytotoxicity. A. HeLa (blue bars), J774A.1 (red bars), or A549 cells (green bars) were infected with the indicated strains at a multiplicity of infection (MOI) of 50 in 24-well plates for 3 h. The bteA mutant strains were complemented in trans with the RB50 bteA allele carried on a medium copy vector (see Methods). Following infection, release of lactate dehydrogenase (LDH) into culture medium was measured as described in Methods. B. bteA homologues were compared using multialign [51] and amino acid differences are shown. Green lines indicate substitutions of highly conserved residues, blue shows weakly similar amino acids, red indicates no similarity, cyan dotted lines designate deletion of a residue and pink designates an amino acid insertion. Bp = B. pertussis, Bpp = B. parapertussis, LRT = lipid raft-targeting domain [12].

Figure 4

Figure 4

In vivo characterization of selected complex IV B. bronchiseptica strains. A. Survival of wild-type female C57BL/6NCr (B6) mice inoculated with different strains of B. bronchiseptica. Groups of four mice were intranasally inoculated with 5 x 105 CFU of the indicated strains in 40 μl volumes as described in Methods. B. Female C57BL/6NCr (B6) mice were infected as above and sacrificed 3 days later. Lungs were removed, homogenized in sterile PBS, and aliquots were plated on selective media. The number of colony forming units (CFU) per lung is shown for each animal. C. Representative H&E-stained sections of lung tissue obtained on day 3 post infection with indicated strains (magnification, x5). D. Histopathological score of indicated strains based on criterion described in Methods. The * indicates P value of <0.0001 for RB50 vs. Bbr77 and RB50 vs. D445.

Figure 5

Figure 5

Comparative genome analysis. A. Cluster analysis of non-core genome sequences of 11 Bordetella strains. The results are displayed using TREEVIEW. Each row corresponds to a specific non-core region of the genome, and columns represent the analyzed strain. Yellow indicates presence while blue represents absence of particular genomic segments. Abbreviations: Bp = B. pertussis, Bpph = human B. parapertussis, Bb IV = complex IV B. bronchiseptica, Bb I = complex I B. bronchisetpica, Bppo = ovine B. parapertussis. B. Zoomed image of non-core region in panel A marked with a red bracket showing complex IV specific regions. On the right, blastn with default settings was used to query the nucleotide collection (nr/nt) from the National Center for Biotechnology Information and homology designations are indicated. C. Distribution of qseBC alleles among complex I and complex IV B. bronchiseptica isolates based on PCR-based amplification and sequencing.

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