Virulence-related characteristics of Bordetella pertussis mutants deficient in intracellular survival

Callie Effler, a student at Lee University, worked in the lab of Dr. Eric Harvill.

Abstract: Intracellular survival is a common trait among human pathogenic bacteria that has advantages for the bacteria’s protection from the host immune response, persistence, and dissemination within the host. Bordetella pertussis, the gram-negative bacteria that causes whooping cough in humans, is commonly regarded as an extracellular pathogen. However, it has been recovered from macrophages in in vitro experiments, and reported anecdotally in clinical samples. It is unknown what contribution to pathogenicity the intracellular population has, if any, on the host. In this work, our broad objective was to evaluate the impact of B. pertussis’ intracellular survival and its role in pathogenicity. To do so, we planned to identify mutants similar to the wild type in general measured aspects of virulence, but that failed to survive inside of macrophages. A transposon library of B. pertussis UT25 was screened, resulting in the identification of several putative mutants that were deficient in intracellular survival. These strains were further screened for intracellular deficiency as a confirmatory measure, and went through further in vitro assays screening for cytotoxicity, hemolytic activity, resistance to serum complementation, and general fitness (growth). Based upon these assays, mutant strain G4 was the best candidate among those tested for an intracellularly-deficient mutant with similar virulence-related characteristics to the wild type. Preliminary C57 mouse infection studies suggest that the mutant strain behaves similarly to the wild type in vivo, indicating that intracellular survival may not be contributing to virulence. It is hypothesized that intracellular survival may be a phenotypic remnant of an ancestral strain of B. pertussis that transitioned from the environment to a become a human pathogen using this trait.


The world’s smallest escape artists: manipulation of the host innate immune response by Bordatella bronchiseptica

Margaret Dedloff, a junior at Clarkson University, worked with Dr. Monical Gestal in the lab of Dr. Eric Harvill to study the role of a sigma factor by bacteria in manipulating host immune response.

Abstract:  During the course of infection, some pathogens are able to sense and respond to the host immune system. This can cause prolonged pathogenesis, high transmission rates, and vaccine failure. One group of bacteria known to manipulate the immune response in varied ways is the Bordetella spp. B. pertussis, B. bronchiseptica and B. parapertussis are able to inhibit complement and suppress B and T-cell functions [1].  The BvgAS two-component system is understood to be a regulator of many virulence factors and is known as the master virulence regulon, however this is most likely an oversimplification. We hypothesized that pathogens like bordetellae should be under strong selective pressure to sense and respond to signals in blood and serum, in order to modulate immune defenses. When studying blood and serum responsive genes, we identified a putative sigma factor up-regulated in both conditions, and we hypothesized that this might be a regulator that dictates adaptation to pressure from the immune system. Here we identified the role of this sigma factor, the Bordetella Sigma Factor, or bsr, in manipulating the immune response. Through the use of a B. bronchiseptica bsr knockout and in vitro assays, we have found that bsr interferes with the innate response. bsr inhibits survival in macrophages by changing the dynamics of phagocytosis, causing macrophage death, and causing bacterial death and replication within the macrophage by interfering with cytokine and chemokine expression as well as differentially interacting with TLR receptors. This suggests that subsequent cell recruitment will be different within the host. Our results demonstrate that the bsr gene plays a critical role in Bordetella interaction with the innate immune system. A better understanding of this gene and its function will be valuable in efforts to create successful vaccines and treatments not only for Bordetella spp. but also for other bacterial species.

[1] Gestal M.C., Whitesides L.T., and Harvill E.T. “Integrated Signaling Pathways Mediate Bordetella Immunomodulation, Persistence & Transmission”. In revision, Current Trends in Microbiology.

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Role of the bsr gene in the Intracellular Survival of Bordetella bronchiseptica

Hira Hasan, a student from Louisiana State University, worked with Monica Cartelle-Gestal in the lab of Dr. Eric Harvell to study the intracellular survival of Bordetella bacteria.

Abstract:  Bordetella pertussis and B. parapertussis are Gram-negative bacteria that cause a respiratory infection, known as whooping cough, in humans. Another member of the Bordetella species, B. bronchiseptica (BB), primarily infects mice, dogs, and horses.  The wild type strain of BB, RB50, contains a gene (bsr) encoding a putative sigma factor that is up-regulated when BB is exposed to blood. To test the role of this gene in pathogen-host interactions, a knock-out mutant called RB50Δbsr was made in our lab. Preliminary results showed that RB50Δbsr survives longer within macrophages than RB50. The mutant also confers sterilizing immunity against further BB, B. pertussis, and B. parapertussis infection in mice, which are excellent models for human infection. The aim of this study was to determine if there is a difference between how RB50 and RB50Δbsr are internalized by macrophages, specifically whether the latter survives longer intracellularly by inhibiting lysosome formation. Confocal microscopy and a lysotracker assay were used to determine the location of bacteria within macrophages, while electron microscopy and several internalization assays were conducted to quantify live bacteria within macrophages overtime. We found that there are higher levels of intracellular RB50Δbsr than RB50 over a 24-hour period, RB50Δbsr does not enter lysosomes readily, and RB50Δbsr infection results in less macrophage death. Based on the results of this study, bsr plays a vital role in macrophage response to BB infection. Since macrophages are involved in activating several other immune system components, manipulating bsr leads to an overall change in the persistence of Bordetella infections.

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