Dr. Keith Mintz
Molecular Mechanisms of Bacterial Pathogenesis
The focus of my research is the identification and characterization of bacterial proteins that are required for tissue colonization, the initiation of any infection. My research interests are concentrated on the membrane proteins and surface structures of the Gram-negative bacterium Aggregatibacter actinomycetemcomitans. A. actinomycetemcomitans is typically associated with Periodontitis, an inflammatory disease of the tissues surrounding and supporting the teeth, which left untreated results in tooth loss. This bacterium is also associated with numerous non-oral diseases including but not restricted to infective endocarditis and atherosclerosis, which suggest that the periodontal pocket is a potential source and reservoir of these diseases.
The emphasis of my research is based on two genes and the associated proteins identified in my laboratory. We have identified and characterized a unique collagen adhesin expressed by A. actinomycetemcomitans. The extracellular matrix protein adhesin A (EmaA) is a 202 kDa protein that trimerizes to form antenna-like structures on the surface of the bacterium. The EmaA structures are required for collagen adhesion. We posit that this adhesin is associated with tropism and colonization of disparate tissues by this pathogen. We have demonstrated that EmaA is important for the colonization of heart valves in an in vitro rabbit explant model and an in vivo rabbit endocarditis model. The collagen binding domain maps to the distal portion of the structure, which corresponds to the amino termini of the monomers. We have also solved the first 3D structure of the functional domain of the adhesin in collaboration with Dr. Teresa Ruiz in the Department of Molecular Physiology and Biophysics at the University of Vermont. We have recently determined that the protein is modified with sugars, which are suggested to be similar to those that compose the O-polysaccharide (O-PS) of the LPS (lipopolysaccharide) of the bacterium. In addition, we have also demonstrated that the posttranslational modification is required for collagen binding activity. The enzymes associated with the biosynthesis of the O-PS are also required for this posttranslational modification. This “cross-talk” between biosynthetic pathways represents a novel glycosylation mechanism for this and other bacteria. We continue to study the posttranslational modifications and structural aspects of the EmaA/collagen interaction. Completion of these studies will permit the dissection of the critical components involved in collagen adhesion and provide new insights into the molecular mechanism of this adhesin.
Bacterial membranes are important for the secretion and presentation of virulence determinants as well as communication between cells. A. actinomycetemcomitans displays an irregular (convoluted) outer membrane morphology. This morphology is associated with the expression of a novel gene, morC (morphogenesis protein C), identified in my laboratory. MorC is a 141 kDa inner membrane protein that is conserved in Gram-negative bacteria. Inactivation of morC results in the loss of membrane convolutions, decreased cell size, increased cell auto-aggregation and increased sensitivity to bile salt. In addition, the absence of this protein abolishes the secretion of leukotoxin, a toxin that specifically targets human white blood cells. The dramatic effect of the loss of MorC attests to the importance of this protein for the physiology of this pathogen. The mechanism by which this critical protein functions is unclear and the long term goal of this arm of my research is to define how MorC and associated proteins determine membrane structure and function in this oral pathogen.
We have completed a quantitative proteomic survey comparing the membrane proteins of the wild type with the morC mutant strain. The study has revealed that the phenotypes displayed by the morC mutant strain are due to changes in the abundance of a small cohort of membrane proteins. We are in the process of inactivating these genes and determining if inactivation of these genes result in a morC-like phenotype. Interestingly, this study also indicated that the amounts of the individual protein components of the leukotoxin secretion apparatus are unchanged when comparing the wild type and mutant strains. This suggests that the absence of MorC results in a conformation change of one or more of the proteins associated with the secretion apparatus, which leads to reduced secretion of the toxin. The same data set has also revealed a decrease in multiple fimbriae secretion apparatus proteins. This suggests that MorC is involved in fimbriae secretion and we have demonstrated the importance of this protein in fimbriae biogenesis and biofilm formation.
322B Stafford Hall
320 & 322 Stafford Hall
Smith KP, Fields JG, Voogt RD, Deng B Lam YW, Mintz KP. Alteration in abundance of specific membrane proteins of Aggregatibacter actinomycetemcomitans is attributed to deletion of the inner membrane protein MorC. Proteomics. 2015 Jun;15(11):1859-67.
Tang G, Mintz KP. Glycosylation of the collagen adhesin EmaA of Aggregatibacter actinomycetemcomitans is dependent upon the lipopolysaccharide biosynthetic pathway. J Bacteriol. 2010 Mar;192(5):1395-404. doi: 10.1128/JB.01453-09.
Yu, C., Mintz, K.P. and Ruiz, T. Investigation of the 3D architecture of the collagen adhesin EmaA of Aggregatibacter actinomycetemcomitans by electron tomography. J Bacteriol. 2009 Oct;191(20):6253-61
Gallant, C.V., Sedic, M., Chicoine, E.A., Ruiz, T. and Mintz, K.P. Membrane morphology and leukotoxin secretion are associated with a novel membrane protein of Aggregatibacter actinomycetemcomitans. J Bacteriol. 2008 Sep;190(17):5972-80.
Tang, G, Kitten, T., Munro, C, Wellman, G. and Mintz, K.P. EmaA, a potential virulence determinant of Aggregatibacter (Actinobacillus) actinomycetemcomitans in infective endocarditis. Infect Immun. 2008 Jun;76(6):2316-24.
Ruiz, T., Lenox, C., Radermacher, M., and Mintz, K.P. Novel surface structures are associated with the adhesion of Actinobacillus actinomycetemcomitans to collagen. Infect Immun. 2006 Nov;74(11):6163-70.