Pglyrp-Regulated Gut Microflora Prevotella falsenii, Parabacteroides distasonis and Bacteroides eggerthii Enhance and Alistipes finegoldii Attenuates Colitis in Mice.

Dysbiosis is a hallmark of inflammatory bowel disease (IBD), but it is unclear which specific intestinal bacteria predispose to and which protect from IBD and how they are regulated. Peptidoglycan recognition proteins (Pglyrps) are antibacterial, participate in maintaining intestinal microflora, and modulate inflammatory responses. Mice deficient in any one of the four Pglyrp genes are more sensitive to dextran sulfate sodium (DSS)-induced colitis, and stools from Pglyrp-deficient mice transferred to wild type (WT) germ-free mice predispose them to much more severe colitis than stools from WT mice. However, the identities of these Pglyrp-regulated bacteria that predispose Pglyrp-deficient mice to colitis or protect WT mice from colitis are not known. Here we identified significant changes in ß-diversity of stool bacteria in Pglyrp-deficient mice compared with WT mice. The most consistent changes in microbiome in all Pglyrp-deficient mice were in Bacteroidales, from which we selected four species, two with increased abundance (Prevotella falsenii and Parabacteroides distasonis) and two with decreased abundance (Bacteroides eggerthii and Alistipes finegoldii). We then gavaged WT mice with stock type strains of these species to test the hypothesis that they predispose to or protect from DSS-induced colitis. P. falsenii, P. distasonis, and B. eggerthii all enhanced DSS-induced colitis in both WT mice with otherwise undisturbed intestinal microflora and in WT mice with antibiotic-depleted intestinal microflora. By contrast, A. finegoldii (which is the most abundant species in WT mice) attenuated DSS-induced colitis both in WT mice with otherwise undisturbed intestinal microflora and in WT mice with antibiotic-depleted intestinal microflora, similar to the colitis protective effect of the entire normal microflora. These results identify P. falsenii, P. distasonis, and B. eggerthii as colitis-promoting species and A. finegoldii as colitis-protective species.

Peptidoglycan recognition proteins kill bacteria by inducing oxidative, thiol, and metal stress.

Mammalian Peptidoglycan Recognition Proteins (PGRPs) are a family of evolutionary conserved bactericidal innate immunity proteins, but the mechanism through which they kill bacteria is unclear. We previously proposed that PGRPs are bactericidal due to induction of reactive oxygen species (ROS), a mechanism of killing that was also postulated, and later refuted, for several bactericidal antibiotics. Here, using whole genome expression arrays, qRT-PCR, and biochemical tests we show that in both Escherichia coli and Bacillus subtilis PGRPs induce a transcriptomic signature characteristic of oxidative stress, as well as correlated biochemical changes. However, induction of ROS was required, but not sufficient for PGRP killing. PGRPs also induced depletion of intracellular thiols and increased cytosolic concentrations of zinc and copper, as evidenced by transcriptome changes and supported by direct measurements. Depletion of thiols and elevated concentrations of metals were also required, but by themselves not sufficient, for bacterial killing. Chemical treatment studies demonstrated that efficient bacterial killing can be recapitulated only by the simultaneous addition of agents leading to production of ROS, depletion of thiols, and elevation of intracellular metal concentrations. These results identify a novel mechanism of bacterial killing by innate immunity proteins, which depends on synergistic effect of oxidative, thiol, and metal stress and differs from bacterial killing by antibiotics. These results offer potential targets for developing new antibacterial agents that would kill antibiotic-resistant bacteria.

Mammalian peptidoglycan recognition proteins kill bacteria by activating two-component systems and modulate microbiome and inflammation.

Peptidoglycan recognition proteins (PGRPs) are conserved from insects to mammals and function in antibacterial immunity. We have revealed a novel mechanism of bacterial killing by innate immune system, in which mammalian PGRPs bind to bacterial cell wall or outer membrane and exploit bacterial stress defense response to kill bacteria. PGRPs enter Gram-positive cell wall at the site of daughter cell separation during cell division. In Bacillus subtilis PGRPs activate the CssR-CssS two-component system that detects and disposes of misfolded proteins exported out of bacterial cells. This activation results in membrane depolarization, production of hydroxyl radicals, and cessation of intracellular peptidoglycan, protein, RNA, and DNA synthesis, which are responsible for bacterial death. PGRPs also bind to the outer membrane in Escherichia coli and activate functionally homologous CpxA-CpxR two-component system, which also results in bacterial death. We excluded other potential bactericidal mechanisms, such as inhibition of extracellular peptidoglycan synthesis, hydrolysis of peptidoglycan, and membrane permeabilization. In vivo, mammalian PGRPs are expressed in polymorphonuclear leukocytes, skin, salivary glands, oral cavity, intestinal tract, eyes, and liver. They control acquisition and maintenance of beneficial normal gut microflora, which protects the host from enhanced inflammation, tissue damage, and colitis.

Peptidoglycan recognition proteins kill bacteria by activating protein-sensing two-component systems.

Mammalian peptidoglycan recognition proteins (PGRPs), similar to antimicrobial lectins, bind the bacterial cell wall and kill bacteria through an unknown mechanism. We show that PGRPs enter the Gram-positive cell wall at the site of daughter cell separation during cell division. In Bacillus subtilis, PGRPs activate the CssR-CssS two-component system that detects and disposes of misfolded proteins that are usually exported out of bacterial cells. This activation results in membrane depolarization, cessation of intracellular peptidoglycan, protein, RNA and DNA synthesis, and production of hydroxyl radicals, which are responsible for bacterial death. PGRPs also bind the outer membrane of Escherichia coli and activate the functionally homologous CpxA-CpxR two-component system, which kills the bacteria. We exclude other potential bactericidal mechanisms, including inhibition of extracellular peptidoglycan synthesis, hydrolysis of peptidoglycan and membrane permeabilization. Thus, we reveal a previously unknown mechanism by which innate immunity proteins that bind the cell wall or outer membrane exploit the bacterial stress defense response to kill bacteria.