Research Projects in the Yoder-Himes Laboratory
Opportunistic infections are those caused by pathogens only in patients with pre-existing conditions, such as immunocompromised patients or diabetes. Pathogens that cause opportunistic infections, by definition, do not infect healthy individuals. Opportunists can be bacterial, viral, algal, or protozoan in nature. Our lab is primarily concerned with opportunistic bacterial pathogens that are found readily in soils and sediments and that can be found in hospital intensive care units where they lead to devastating and extremely difficult to treat infections. These organisms also cause pneumonia in people with cystic fibrosis.
Cystic Fibrosis (CF) is the most common lethal genetic disorder in Caucasian populations with an incidence of 1 in every 2500 live births in the U.S. alone. It is caused by a autosomal recessive genetic mutation in the CFTR gene which leads to a salt imbalance in the mucous membranes in the human body including the lungs and the intestines. This salt imbalance leads to the production of sticky mucous to be produced which is prime breeding grounds for bacteria. Due to the number of bacteria that infect the lungs over the course of CF patient’s lives, the mean survival age of CF patients is ~45 years, up from 35 years in the last decade due to medical advances.
The main pathogens that infect the lungs of CF patients are Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, Stenotrophomonas maltophilia, and members of the Burkholderia cepacia complex, as well as a handful of others. While P. aeruginosa is by far the most common, it is also usually the first to infect CF patients and it can be relatively easy to treat despite its natural ability to resist multiple antibiotics. It is also rarely passed patient-to-patient, thus it is easily contained. However, P. aeruginosa, like S. maltophilia, and the B. cepacia complex, is readily isolated from soils across the globe; thus, the threat of re-infection of CF patients, cannot be eradicated.
While the rarest of the five common pathogens is B. cepacia complex (Bcc), this is a particularly interesting group of organisms. Comprised of ≥17 closely related species, this group is environmentally ubiquitous and can survive in numerous niches, including waters, on plastics, in cosmetics, and in normally sterile solutions such as mouthwashes and disinfectants. Like P. aeruginosa, the Bcc are very antibiotic resistant and have a high GC content in their genomes, though these two groups are relatively distantly related among all bacteria. The Bcc can lead to a rapid necrotizing pneumonia, known as “cepacia syndrome” which is characterized by a fast decline in lung function over the space of 1-3 months and always results in mortality once the bacteria enter the bloodstream.
Certain species within the Bcc have been passed patient-to-patient and have caused epidemics in CF clinics in the U.S., Canada, and Europe. Strict regulations have been instituted world-wide for CF patients that are colonized with Bcc members in which patients are isolated from the general CF population in order to minimize the spread. Burkholderia cenocepacia is the most common organism to cause epidemics while others, such as B. multivorans, rarely do. One of the latest epidemics occurred at Children’s Hospital Boston in which Burkholderia dolosa (one of the Bcc members) infected over 40 patients and led to the death of over 7 patients by cepacia syndrome.
The Bcc is also interesting because it is closely related to Burkholderia pseudomallei and Burkholderia mallei which are the causative agents of melioidosis and glanders in humans and horses, respectively. Both are considered Select Agents by the U.S. government and are listed as Biosafety Level 3 which requires containment to research. These organisms were reportedly used as biowarfare during WWI and WWII in attempts to poison U.S. horses. Jonathan Warawa at the University of Louisville Medical School has an active program studying B. pseudomallei and B. mallei.
More information on CF and its opportunistic pathogens can be found at the following websites:
Our long term goal is to investigate the evolution of virulence properties, transmission from the environment to humans, and the ecology of opportunistic bacterial respiratory pathogens in humans and the environment.
To achieve these goals, we have three projects currently in the lab.
Comparative transcriptomics in polymicrobial cultures
While we now understand the messages made by bacteria when they are alone, we generally don’t know what they are saying when in the presence of other bacteria. And most of the time in any environment, they are surrounded by other species. For example, there are many different bacteria co-habitating in the human gut or in the soil. Are they fighting? Are they forming collaborations? Are they ignoring each other? Why are only some types of bacteria found in certain environments and not others? For example, why don't we find Streptococcus pneumoniae, a causative agent of pnuemonia in humans, not found in cystic fibrosis lungs or very commonly in soils?
In any environment, the bacteria are competing for nutrients, space, and other resources so it makes sense to know who your neighbors are. Are they nice neighbors or are they jerks who will kill you as soon as look at you? You need to know these things so you can adapt and prepare to defend your space.
So we need to know what they are saying and how they sense their neighbors. To answer this, we can use next-generation techniques like RNA-seq and Tn-seq to address those genes that are expressed or are essential for survival under a given set of conditions, whether it is a bacterial strain alone in a culture or in a mixed biofilm with other species. We can compare the differences between bacteria in liquids (like in growth medium) or on surfaces (plastic plates or tissue culture cells), which is more similar to how they live in the natural environments, whether it is the human lung or the soil.
This project is good for those students who would like to learn some of the newest techniques that are starting to dominate the field of molecular biology. It also requires some ability to use bioinformatics and would be very good for anyone wanting to increase their marketability, especially if you are willing to learn some computer programming.
To define and pursue Burkholderia cepacia complex virulence factors and potential vaccine targets
This is especially necessary as antibiotic resistance in this group of bacteria rises. The goal of one part of this project is to refine the 126 B. cenocepacia virulence factors and 8 putative therapeutic targets I have already identified (Yoder-Himes et al. PNAS 2009) by performing RNA-seq on additional clinical Bcc strains (e.g. B. multivorans,. B. dolosa) and finding genes whose expression is induced preferentially under CF-like conditions or in host systems. By comparing with my previous data sets, we can further refine our list of putative virulence factors to those that are expressed under host-like conditions in many Bcc species. A second goal is to further characterize the genes in B. dolosa essential for survival in a mouse. These are genes I have identified using Tn-seq. These particular candidates are interesting because we know they are expressed in a host system and therefore may be good targets for antibiotics. Finally, a third goal would be to follow up the putative vaccine targets by purifying them following overexpression in a heterologous system and testing their immunogenic properties in tissue cultures, transcytosis assays, and in mice. The results of this work may also provide therapeutics for the related Select Agents B. pseudomallei and B. mallei and thus have biodefense applications.
This project is good for students who are interested in classic molecular biology in bacterial pathogenesis. The techniques learned here will be applicable to many projects over the course of a student’s career and are the fundamental tasks that students should know.
Testing environmental, particularly airborne, Burkholderia and Pseudomonas strains for biofilm properties and gene expression;
A longer-term goal and my passion is to study the transmission of these pathogens to humans. To do this, we will sample surface and air populations in environmental and clinical locations, identify the organisms in the air using culture-independent techniques, culture the Pseudomonas and Burkholderia populations using general and selective media, and determine if these strains can form biofilms and contain newly identified virulence genes. In addition, I would like to ask if the bacteria in the air are actively responding to this ecological niche by isolating RNA from air-borne microorganisms and sequencing the corresponding cDNA.
This project would be great for undergraduates in classes to take on as it would involve field work and data analysis leading to publications.