| Since B. anthracis enters a host animal as a metabolically dormant endospore and disease pathology is mediated by vegetative bacilli, I hypothesize that differentiation between the two is vital to disease establishment. To test this hypothesis, I introduce specific lesions into the B. anthracis genome, altering various aspects of germination and outgrowth, the processes that converts an endospore to a vegetative cell. Mutants defective at both regulatory and biochemical stages were analyzed in vitro and in vivo. Several mutants were strikingly less pathogenic than their parental strain.; Germinant receptors within the endospore constantly monitor the cell's microenvironment and initiate germination upon recognition of host molecular signals. This recognition step was evaluated through construction of endospores devoid of each germinant receptor or of coat proteins encoded by the gerP operon that control access to all germinant receptors. Five germinant receptors influence in vitro germination or animal virulence, and cognate signals were identified for all but one. Correlation between the in vitro loss of signal sensory capabilities and virulence suggests B. anthracis recognizes its hosts simultaneously sensing multiple chemical signals, all of which may exist in the environment-at-large, albeit primarily in the absence of others.; After host recognition, morphological differentiation requires coordinated biochemistries of at least two types: hardened structures of the endospore must be degraded and vegetative ultrastructures must be synthesized anew. Mutation of sleB slowed degradation of the endospore-specific cell wall, and resulted in a significant decrease in virulence. These data suggest cellular differentiation is exquisitely linked to pathogenesis such that either premature, delayed, or prolonged germination is grossly attenuating.; Furthermore, B. anthracis cells must execute this differentiation process while combating host immunity. I found the dltABCD operon to be expressed immediately following the onset of germination, and throughout outgrowth and vegetative growth. The protein products of this operon are responsible for D-alanyl esterification of a vegetative cell wall component, a function central to bacterial resistance against peptide, enzymatic, and cellular mediators of innate immunity. Loss of dltABCD expression also decreased B. anthracis lethality. These findings allow a preliminary model of early B. anthracis-host interactions to be presented. |
