Novel Mechanisms of Bordetella Survival in the Mammalian Host and in the Environment

Open Access
Author:
Bendor, Liron
Graduate Program:
Genetics
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
September 04, 2015
Committee Members:
  • Eric Thomas Harvill, Dissertation Advisor
  • Robert Paulson, Committee Chair
  • Kenneth Charles Keiler, Committee Member
  • Mary J Kennett, Committee Member
  • Ross Cameron Hardison, Committee Member
Keywords:
  • Bordetella
  • bordetella bronchiseptica
  • amoeba
  • dictyostelium
  • Type Six Secretion System
  • intracellular survival
Abstract:
The Bordetella are respiratory pathogens that infect a wide variety of hosts, and this dissertation focuses on novel mechanisms employed by these bacteria to survive in the mammalian host and in the environment. We highlight in chapter one the mechanisms used by Bordetella species for pathogenesis and survival during infection, and review immune responses to bacterial infection. Chapter two describes a novel role for the Type Six Secretion System during infection. An analysis of 58 recently sequenced B. bronchiseptica isolates revealed that one-third of recently sequenced Bordetella bronchiseptica strains of the predominantly human-associated Complex IV have lost their T6SS through gene deletion or degradation. Since the majority of B. bronchiseptica human infections occur in immunocompromised patients, loss of the T6SS may be selectively advantageous for B. bronchiseptica survival in immunocompromised hosts. Infection of immunodeficient mice (Rag1-/-) with a T6SS-deficient mutant of B. bronchiseptica caused hyperlethality as compared to infection with the wild-type strain. Additionally, large numbers of the T6SS-deficient mutant (but not wild-type) bacteria were recovered from systemic organs 21 days post-inoculation. While the T6SS was shown to decrease pathology and pro-inflammatory cytokine production during infection of Rag1-/- mice, co-infection of the wild-type and T6SS-deficient strains revealed that a heightened inflammatory response associated with infection of the T6SS-mutant is not sufficient to enable B. bronchiseptica translocation to systemic organs. Rather, the T6SS-deficient B. bronchiseptica strain survives in Antigen Presenting Cells (APCs) after phagocytosis and is trafficked to systemic organs inside these cells. Meanwhile, wild-type B. bronchiseptica kills its eukaryotic cellular host via a T6SS-dependent mechanism and is thus not recovered systemically in immunodeficient mice. We concluded that the bacterial T6SS and host adaptive immune components both limit B. bronchiseptica survival intracellularly during infection. Loss of the T6SS in recent B. bronchiseptica isolates, therefore, may enable persistence in an intracellular niche in immunocompromised human patients. In chapter three, amoebae are identified as potential environmental reservoirs for the bordetellae. We showed that B. bronchiseptica efficiently infects single-celled Dictyostelium discoideum trophozoites and survives and replicates in sori of D. discoideum multi-cellular fruiting bodies. Additionally, the enigmatic Bvg- phase is identified as important for survival in amoeba sori. While several other bacterial species have been shown to survive endosymbiotically in amoeba sori, we show that B. bronchiseptica lowers amoeba spore numbers over time while replicating in sori and thus hijacks the amoeba mechanism of dissemination for its own benefit. By passaging B. bronchiseptica-containing sori onto lawns of Klebsiella aerogenes, (an amoeba food source), we identified a stable relationship between B. bronchiseptica and D. discoideum such that the bacteria can be recovered from the sorus through multiple amoebic lifecycles. While in amoeba sori, B. bronchiseptica can be geographically disseminated by flies and can efficiently infect mice. These results suggest that amoebae act as an environmental reservoir, replication medium, and transmission vector for B. bronchiseptica. We continue to examine B. bronchiseptica – amoebic interactions in chapter four. While most amoeba resistant bacteria (ARBs) prevent D. discoideum differentiation into fruiting bodies, B. bronchiseptica does not prevent D. discoideum initial plaque and fruiting body formation but rather inhibits plaque expansion. In contrast, amoeba plaques formed on lawns of K. aerogenes expand efficiently over time. Additionally, we identify the adenylate cyclase toxin as important for B. bronchiseptica inhibition of plaque expansion. Interestingly, B. bronchiseptica does not inhibit amoeba plaque growth when mixed with even low levels of K. aerogenes, suggesting that amoebae circumvent the B. bronchiseptica mechanism of inhibition when other bacterial species are present. However, growth of plaques and fruiting bodies on mixed B. bronchiseptica and K. aerogenes lawns are altered, as evidenced by decreased fruiting body production relative to those formed on K. aerogenes alone. Therefore, in the presence of other bacterial species B. bronchiseptica loses its ability to inhibit plaque expansion but is still harmful to amoebae. Importantly, B. bronchiseptica is recovered from sori of amoebae grown on mixed B. bronchiseptica and K. aerogenes lawns. These data suggest that B. bronchiseptica has a selective advantage as compared to bacterial “food” species in the presence of amoebae due to its evolved ability to survive predation and replicate in amoeba sori. This dissertation highlights novel mechanisms employed by Bordetella for survival in mammalian hosts and in the environment. It establishes that the T6SS limits intracellular survival and that the loss of the T6SS in B. bronchiseptica strains may be linked to B. bronchiseptica survival in the human population. Furthermore, it identifies amoeba as a potential environmental niche for the bordetellae and elucidates the role of the B. bronchiseptica Bvg- phase for survival in this niche. Lastly, this work delves into the complex interactions that enable B. bronchiseptica to avoid predation by and utilize D. discoideum in geographical dissemination and transmission to the next host. Altogether, this dissertation contributes to our understanding of B. bronchiseptica transmission, pathogenicity, and persistence in novel mammalian and environmental niches.