Margaret McFall-Ngai & Edward (Ned) Ruby

About our Lab

Our research focuses on host responses to interactions with beneficial microbes. Within this context, the studies of my laboratory address five major questions:

  1. How are environmentally rare bacteria harvested from the host’s habitat during the onset of a horizontally transmitted symbiosis?
  2. By what mechanisms does the host recognize its specific symbiotic partner(s)?
  3. What are the influences of symbiotic bacteria on the developmental of the host tissues with which they associate?
  4. How is the symbiont population maintained in balance over the host’s lifetime, such that neither does the symbiont overgrow the host nor does the host eliminate the symbiont?
  5. What are the similarities and differences between pathogenic and beneficial animal-bacterial interactions?

Essentially all animal and plant species have long-term associations with specific microbial symbionts. In many cases, these associations have been shown to be essential to the normal health and development of the hosts. Among the most common kinds of interactions present in animals are those between extracellular bacteria and epithelial tissue. The long-term objectives of our research are to define the behavioral, physiological and molecular events that characterize the bacterial colonization of animal epithelial tissue. Our specific aims are to:
  1. Determine how specificity is achieved in environmentally transmitted bacteria-host associations. 
  2. Identify aspects of bacterial behavior that are essential to tissue tropism. 
  3. Characterize the molecular mechanisms that underlie successful tissue colonization. 
  4. Comparatively examine virulence factors of pathogenic bacteria that are required for symbiotic colonization. 
  5. Define the spatial patterns and temporal dynamics of non-clonal bacterial populations within monospecific associations. 
  6. Determine the suite of changes in bacterial gene expression that are induced by the environment of host tissues.

Approaches

squidWe use the squid-vibrio (Euprymna scolopes-Vibrio fischeri) light organ system as a model to approach these broad questions. Over the past ten years of our development of this system, we have focused on defining the anatomical and biochemical influences of the microbial symbiont on the host, using standard techniques, including confocal, electron and light microscopy and routine protein biochemistry. In recent years, we have added to our approaches the role of symbionts in the induction of host gene expression. To this end, in collaboration with the laboratories of Drs. Bento Soares and Thomas Casavant of the University of Iowa, we have developed a unigene set from the cDNA libraries of the host squid light organ, which contains nearly 14,000 unique genes. We are now launched into RNAseq technology to describe the changes in host gene expression with symbiosis.

Approaches

squidTo accomplish these goals we have chosen as a natural study system the symbiotic association of the light-emitting organ of the squid Euprymna scolopes by the luminous bacterium Vibrio fischeri. This system provides a simple and experimentally accessible paradigm for studying specific host-bacterial interactions. Our recent investigations have centered on the events characterizing the initiation, colonization, and persistence of the symbiotic infection in newly hatched juvenile squids using bacterial mutants to manipulate, assay, and observe the results of the complex program of signaling and responses through which the host and bacterium communicate. We believe that this system also serves as a model of infection by pathogenic species of Vibrio and, perhaps, of the evolution of the virulence state in these and other pathogens; that is, bacterial and host determinants that potentiate light-organ symbiosis are revealing convergences with known bacterial virulence factors, and are promoting the discovery of as yet undescribed ones. This work will also aid our understanding of the mechanisms by which the benign colonization of mollusk tissue may serve as a reservoir for human pathogenic Vibrio species.

VibrioIn recent years we, in conjunction with other labs working with Vibrio fischeri and the squid host, have developed and applied novel technical approaches to facilitate our research aims. Specifically, we have developed molecular genetics in this bacterium, sequenced the genome of a light organ isolate, and created transposon mutant libraries and glass-chip and Affymetrix DNA microarrays. In addition, using fluorescently labeled V. fischeri cells and confocal microscopy, we are characterizing the behavior of these bacteria during the initiation and development of symbiosis in living squid. Finally, by adapting microinjection techniques, we have begun to introduce symbiotically incompetent bacteria into the light organ to determine the colonization stage in which they are defective.

Findings

Using these techniques, our principal findings with respect to the above-mentioned questions have been as follows:
  1. We have described the mechanism by which the newly hatched host enriches for V. fischeri, which accounts for less than 0.1% of the bacteria in the surrounding seawater. Non-specific, environmental peptidoglycan (a cell surface component of all bacteria) induces the host to shed mucus from an epithelial field near the site of colonization. V. fischeri specifically aggregates in this mucus for several hours and then invades host tissues. 

  2. cryptsWe have found that specificity begins outside the sites of eventual colonization, i.e., in the host-shed mucus. Further specificity determinants include: the provision of high levels of nitric oxide, a toxic oxygen species, in the ducts through which symbionts must migrate to colonize host crypt spaces; mannose-glycan interactions between the symbiont surfaces and host crypt epithelial cells. 

  3. We have documented that V. fischeri induces profound developmental effects on the host light organ tissues, including apoptosis, macrophage-infiltration, cell swelling, increase in microvillar density, and a total remodeling of light organ morphology. Much of this development is mediated through bacterial factors, most commonly known as ‘microbe associated molecular patterns’ or ‘MAMPs’. Specifically, components of symbiont surface molecules, lipopolysaccharide and peptidoglycan, interact with host crypt epithelial to induce developmental pathways. The specific peptidoglycan fragment was identified as ‘tracheal cytotoxin’ or TCT, a molecule that has thus far only been reported to be exported by two pathogens, Neisseria gonorrheae and Bordetella pertussis

  4. squidWe have found that the symbiosis is controlled by a dramatic diel rhythm. Each day at dawn, the host vents 90-95% of its symbiont culture into the surrounding environment. Coupled with this rhythm is a marked remodeling of the crypt tissues. After venting, the tissues organize into highly polarized epithelia with dense lobate microvillar surfaces. With progression over the day, the tissues begin to take on a stressed appearance, with effacement of the epithelia and blebbing of the apical surfaces of the epithelial cells. Just before venting, the tissue is highly disorganized. 

  5. The finding that ‘MAMPs’ mediate harvesting and much of development in the squid-vibrio system suggests that, at least in this system, beneficial and pathogenic bacteria are likely to use a similar molecular language to foster interaction.
 

Current Research Directions

  1. Mutagenesis Library: Construction of an ordered mutant library of the bacterial symbiont, Vibrio fischeri
  2. Flagella in Accommodation and Persistence: Investigating the role of flagella in the later stages of colonization of the light organ using microinjection techniques. 
  3. Competitive Dominance: The earliest site of species specificity in the V. fischeri-E. scolopes partnership is the dominance of V. fischeri in the host-derived mucus. 
  4. Natural Competence: Investigating the relationship between chitin and extracellular DNA uptake and use in V. fischeri
  5. AinS-mediated Quorum Sensing: Determining the role of the Vibrio fischeri AinS quorum-sensing system in the colonization of Euprymna scolopes
  6. Bacterial Diversity: Can static and dynamic patterns of diversity existing in natural V. fischeri populations found in E. scolopes light organs be related to current ecological and evolutionary theory? 
  7. Nitric Oxide Stress in the light organ environment: Study on the mechanisms of how Vibrio fischeri responds to and survives NO stress during symbiotic colonization to Euprymna scolopes.

Future Directions

Our immediate goals are to use the information that is becoming available through bioinformatics to deepen our understanding of the interaction between the host and symbiont in the squid-vibrio association. These data will provide heuristic value for the continued study of the above-described aims. However, although bioinformatics will be brought into the study of this system, the conceptual focus will always remain at the more organismic level, i.e., understanding how two species, from very different phylogenetic origins, manage to form a persistent relationship.

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Margaret McFall-Ngai & Edward Ruby

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McFall-Ngai Lab
Margaret McFall-Ngai
Phone: (808) 539-7331
E-mail: mcfallng@hawaii.edu

Ruby Lab
Edward (Ned) Ruby
Phone: 808-539-7300
E-mail: eruby@hawaii.edu