The Michael Malamy Lab
I maintain an active research laboratory but no longer accept PhD students.
Members of the genus Bacteroides, are classified as obligate anaerobes, and comprise more than 50% of the total bacteria found in the human large intestine. These commensal bacteria play an important role in human nutrition and in preventing the establishment of potentially pathogenic bacteria in the colon. We are studying B. fragilis, a minority component of the colonic Bacteroides species, but an opportunistic pathogen if released from the large intestine into otherwise sterile cavities of the host where it contributes to abscess formation. In order to emerge as the predominant Bacteroides species to be isolated from sites of infection, B. fragilis must contain a number of virulence factors that confer specific advantages to this organism. Our major goal in the recent past has been to set up genetic systems that allow us to determine what these factors are and to manipulate them to test for alterations of pathogenicity in animal models of infection.
We have concentrated our work on understanding many of these virulence factors including:
Aerotolerance
Although unable to grow in the presence of high concentrations of oxygen in normal atmosphere or in well aerated tissues, and blood, B. fragilis is aerotolerant; it is not killed by exposure to these oxygen levels. How does B. fragilis protect itself from this high oxygen level. Recently, we have discovered a class of B. fragilis mutants that can now grow in the presence of 0.5-1% oxygen, levels that are considered to be microaerophilis. All of the mutants have a defect in the same gene which we have named oxe, for oxygen enabled. The questions then are: why doesn’t B. fragilis grow in the presence of high concentrations of oxygen? What is altered in the oxe mutants so that they now grow with 1% oxygen?
Nutrient Acquisition
B. fragilis seems to be a professional nutrient scavenger with the ability to obtain carbon and nitrogen sources from peptides, glycoproteins, and polysaccharides. Although the colon can be considered to be a rich nutrient environment, we have shown that mutants deficient in hexose utilization are unable to compete with wild-type organisms when tested in a mouse model of colonization. Other experiments reveal that B. fragilis and some other related Bacteroides species possess a sialic acid N-acetyl neuraminic acid (aeaA) utilization pathway that is distinct from that found in many other bacteria. This pathway likely confers a competitive selective advantage to Bacteroides in the colon and at other sites of infection.
Figure 1. Comparison of the NANA and sugar utilization pathways found in E. coli (left) and B. fragilis (right). More details can be found in Brigham et al 2009.
Current experiments will test whether or not manipulation of the animal’s diet can be used to select for or against mutants with defects in utilization of specific substrates. We plan to extend these experiments to the germ-free mouse to better control the influence of other members of the colonic flora in colonization.
Antiobiotic Resistance and Transfer
We pioneered the investigation of drug resistance transfer in B. fragilis and have discovered a number of transposable elements, some of which confer conjugation ability, and have characterized several conjugal and mobilizable elements in this organism. This work lead to the development of genetic systems to analyze virulence factors, including cloning vectors, shuttle vectors, promoter traps, etc.
Anaerobic Respiration
Many Bacteroides species including B. fragilis, B. thetaiotaomicron and B. uniformis have electron transfer chains that greatly increase their yields of ATP per mole of glucose to power all cellular functions. This is mainly accomplished by NADH oxidases that couple the oxidation of NADH to NAD and simultaneously export sodium ions or protons from the inside of the cell to the periplasm thus establishing ion gradients that power such processes as transport of nutrients into the cell, interact with membrane ATPases to synthesize ATP, etc. My lab had previously discovered that B. fragilis can even use low levels of oxygen as the terminal electron acceptor using the cytochrome BD oxidase. Thus B. fragilis is not a strict anaerobe but instead is a nanaerobe! Together with Blancas Barquera at RPI and Laurie Comstock at U. Chicago, we are defining the biochemistry, genetics and in vivo (gnotobiotic mice) functions of these electron transfer systems.
Role of Anaerobes in Evolution
The anaerobes are ancient, having appeared on Earth more than 2.5 billion years ago when the atmosphere and seas was devoid of measurable oxygen. Significant concentrations of oxygen began to accumulate in the atmosphere when the cyanobacteria learned to split water using solar energy. The simple eucaryotes followed but current oxygen levels were not achieved until the advent of the green plants. The anaerobes must have been restricted to oxygen depleted niches and only much later became associated with higher eucaryotes in compartments that could be oxygen free, such as the distal portions of the intestines. It is possible that the anaerobes are biochemically living fossils and the elucidation of their pathways for macromolecular synthesis and their components might illuminate the evolutionary history of these pathways. As an example, we have found that the pathway biosynthesis of the amino acid arginine in B. fragilis and its close relatives is unique, using substrates that are not found in this pathway in other bacteria or higher forms. Interestingly, the aconitase enzyme of B. fragilis is most closely related to the aconitase found in the mitochondria of higher eucaryotes, rather than the aconitase found in E. coli and most other bacteria. Did the eucaryotic mitochondrial enzyme arise from the “swallowing” of primitive bacteria such as B. fragilis? There are several other proteins in B. fragilis that are most closely related to their counterparts in higher eucaryotes. Are these also examples of the bacterial origin of eucaryotic organelles? We continue to discover examples of unique biochemistry in B. fragilis and hope that our work will contribute to understanding the development of these pathways from the beginning of life on Earth.