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. 2013 Nov;12(11):3036-51.
doi: 10.1074/mcp.M113.029041. Epub 2013 Jun 24.

Novel Burkholderia Mallei Virulence Factors Linked to Specific Host-Pathogen Protein Interactions

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Free PMC article

Novel Burkholderia Mallei Virulence Factors Linked to Specific Host-Pathogen Protein Interactions

Vesna Memisević et al. Mol Cell Proteomics. .
Free PMC article

Abstract

Burkholderia mallei is an infectious intracellular pathogen whose virulence and resistance to antibiotics makes it a potential bioterrorism agent. Given its genetic origin as a commensal soil organism, it is equipped with an extensive and varied set of adapted mechanisms to cope with and modulate host-cell environments. One essential virulence mechanism constitutes the specialized secretion systems that are designed to penetrate host-cell membranes and insert pathogen proteins directly into the host cell's cytosol. However, the secretion systems' proteins and, in particular, their host targets are largely uncharacterized. Here, we used a combined in silico, in vitro, and in vivo approach to identify B. mallei proteins required for pathogenicity. We used bioinformatics tools, including orthology detection and ab initio predictions of secretion system proteins, as well as published experimental Burkholderia data to initially select a small number of proteins as putative virulence factors. We then used yeast two-hybrid assays against normalized whole human and whole murine proteome libraries to detect and identify interactions among each of these bacterial proteins and host proteins. Analysis of such interactions provided both verification of known virulence factors and identification of three new putative virulence proteins. We successfully created insertion mutants for each of these three proteins using the virulent B. mallei ATCC 23344 strain. We exposed BALB/c mice to mutant strains and the wild-type strain in an aerosol challenge model using lethal B. mallei doses. In each set of experiments, mice exposed to mutant strains survived for the 21-day duration of the experiment, whereas mice exposed to the wild-type strain rapidly died. Given their in vivo role in pathogenicity, and based on the yeast two-hybrid interaction data, these results point to the importance of these pathogen proteins in modulating host ubiquitination pathways, phagosomal escape, and actin-cytoskeleton rearrangement processes.

Conflict of interest statement

Conflict of interest: The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The combined in silico, in vitro, and in vivo approach. We used bioinformatics tools to identify 49 proteins putatively involved in virulence. These proteins were then screened for interaction partners using yeast two-hybrid (Y2H) assays against whole human and whole murine proteome libraries. We mapped the identified host-pathogen protein-protein interactions (PPIs) to host pathways and host PPI networks. The host proteins interacting with Burkholderia mallei proteins were characterized with respect to their functions and possible role in pathogenicity. This analysis resulted in the identification of a three promising virulence factor candidates. We verified that mutants lacking these proteins showed attenuated virulence in a mouse aerosol challenge model. Finally, we used all obtained data to delineate mechanisms of pathogenicity and generated hypotheses about the potential roles of these three proteins in B. mallei invasion of the host cell.
Fig. 2.
Fig. 2.
Y2H host-pathogen protein–protein interactions. Our yeast two-hybrid (Y2H) assays against whole human and murine proteome libraries identified 600 protein–protein interactions (PPIs) between 21 B. mallei proteins and 409 human proteins (A) and 846 PPIs between 24 B. mallei proteins and 574 murine proteins (B). Green nodes represent B. mallei proteins, whereas purple and red nodes represent host proteins. There are 33 conserved interactions between these two data sets (red edges), i.e. in 33 PPIs, human proteins (red nodes in A) interacted with the same B. mallei proteins as their murine orthologs (red nodes in B).
Fig. 3.
Fig. 3.
Mouse aerosol challenge model results. In the first set of experiments, 40 BALB/c mice (10 mice for each of the three B. mallei mutants + 10 mice exposed to the wild-type strain) received inhalation doses of ≥2 × 104 colony-forming units (cfu), corresponding to ≥10 LD50 for the wild-type strain. In the second set of experiments, additional 40 BALB/c mice received inhalation doses of ≥2 × 105 cfu, corresponding to ≥20 LD50 for the wild-type strain. In both experiments, animals were monitored for 21 days. At the end of the first set of experiments, all 30 mice exposed to mutant strains survived 21 days post-exposure (pink, orange, and blue lines), whereas seven mice exposed to the wild-type strain did not survive (green line). At the end of the second set of experiments, all 30 mice exposed to mutant strains also survived 21 days post-exposure (pink, orange, and blue lines), whereas all 10 mice infected with B. mallei ATCC 23344 died (purple line). There was a statistically significant difference in the survival rate of mice exposed to mutant strains and mice exposed to the wild-type strain in both sets of experiments (p = 4 × 10−3 and p = 9 × 10−5 for the first and second set of experiments, respectively). These results support the notion that each of the three mutants attenuates virulence when infected in mice via the inhalational route of infection.
Fig. 4.
Fig. 4.
Host-pathogen interactions for three B. mallei virulence factors. Three B. mallei virulence factors (green nodes) interact with 192 human (A) and 236 murine (B) proteins (purple and red nodes). There are 12 conserved protein-protein interactions (PPIs) between these two PPI data sets (red edges), i.e. in 12 PPIs, human proteins (red nodes in A) interacted with the same B. mallei proteins as their murine orthologs (red nodes in B). The host proteins (human/murine) in the conserved interactions include: activating signal cointegrator 1 complex subunit 3 (ASCC3/Ascc3), epididymal secretory protein E1 (NPC2/Npc2), sperm-associated antigen 17 (SPAG17/Spag17), ARCN1 protein-coatomer subunit-δ (ARCN1/Arcn1), UMP-cytidine monophosphate kinase (CMPK1/Cmpk1), heat shock 70-kDa protein 5 (HSPA5/Hspa5), bisphosphoglycerate mutase (BPGM/Bpgm), DnaJ homolog subfamily B, member 4 (DNAJB4/Dnajb4), syntaxin-8 (STX8/Stx8), and thioredoxin-like protein 1 (TXNL1/Txnl1).

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