Comparative genomics investigations have yielded an abundance of information on the genetic variation between organisms, however understanding the consequences of this variation for protein function has proved challenging. We present a profile HMM-based method for assessing the functional significance of mutations in protein coding sequences. We demonstrate an application of the method to comparative analysis of bacterial genomes to scan for functionally significant genetic variation. As a model system, we have chosen the well-studied species Salmonella enterica, of which several lineages are known to have undergone adaptation to an invasive infection style, associated with a narrowing of host tropism with a concurrent accumulation of pseudogenes. We show that our method is able to detect functional degradation of genes associated with host-adapation that are not detected by conventional pseudogene analysis, and suggest that this approach offers a sensitive measure of the loss-of-function mutations that may occur as a result of adaptation to a new niche.