Insects depend solely upon innate immune responses to survive infection. These responses include the activation of extracellular protease cascades, leading to melanization and clotting, and intracellular signal transduction pathways inducing antimicrobial peptide gene expression. In Drosophila, the IMD pathway is required for antimicrobial gene expression in response to gram-negative bacteria. The exact molecular component(s) from these bacteria that activate the IMD pathway remain controversial. We found that highly purified LPS did not stimulate the IMD pathway. However, lipid A, the active portion of LPS in mammals, activated melanization in the silkworm Bombyx morii. On the other hand, the IMD pathway was remarkably sensitive to polymeric and monomeric gram-negative peptidoglycan. Recognition of peptidoglycan required the stem-peptide sequence specific to gram-negative peptidoglycan and the receptor PGRP-LC. Recognition of monomeric and polymeric peptidoglycan required different PGRP-LC splice isoforms, while lipid A recognition required an unidentified soluble factor in the hemolymph of Bombyx morii.

Recent studies of peptidoglycan recognition protein (PGRP) have shown that 2 of the 13 Drosophila PGRP genes encode proteins that function as receptors mediating immune responses to bacteria. We show here that another member, PGRP-SC1B, has a totally different function because it has enzymatic activity and thereby can degrade peptidoglycan. A mass spectrometric analysis of the cleavage products demonstrates that the enzyme hydrolyzes the lactylamide bond between the glycan strand and the cross-linking peptides. This result assigns the protein as an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), and the corresponding gene is thus the first of this class to be described from a eukaryotic organism. Mutant forms of PGRP-SC1B lacking a potential zinc ligand are enzymatically inactive but retain their peptidoglycan affinity. The immunostimulatory properties of PGRP-SC1B-degraded peptidoglycan are much reduced. This is in striking contrast to lysozyme-digested peptidoglycan, which retains most of its elicitor activity. This points toward a scavenger function for PGRP-SC1B. Furthermore, a sequence homology comparison with phage T7 lysozyme, also an N-acetylmuramoyl-L-alanine amidase, shows that as many as six of the Drosophila PGRPs could belong to this class of proteins.

Blood is arguably the most important bodily fluid and its analysis provides crucial health status information. A first routine measure to narrow down diagnosis in clinical practice is the differential blood count, determining the frequency of all major blood cells. What is lacking to advance initial blood diagnostics is an unbiased and quick functional assessment of blood that can narrow down the diagnosis and generate specific hypotheses. To address this need, we introduce the continuous, cell-by-cell morpho-rheological (MORE) analysis of diluted whole blood, without labeling, enrichment or separation, at rates of 1000 cells/sec. In a drop of blood we can identify all major blood cells and characterize their pathological changes in several disease conditions in vitro and in patient samples. This approach takes previous results of mechanical studies on specifically isolated blood cells to the level of application directly in blood and adds a functional dimension to conventional blood analysis.

Background: The regulation of cell wall hydrolysis by the pneumococcal autolysin LytA is poorly understood.Results: The cell wall is susceptible to extracellular LytA only during the stationary phase or after cell wall synthesis inhibition.Conclusion: LytA is regulated on the substrate level, where peptidoglycan modifications likely prevent LytA hydrolysis.Significance: The control of amidases is essential for bacterial survival, cell-wall synthesis, and division.