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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 β-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.

Mammalian Peptidoglycan Recognition Proteins (PGRPs), similar to antimicrobial lectins, bind to bacterial cell wall and kill bacteria through an unknown mechanism. We show that 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, cessation of intracellular peptidoglycan, protein, RNA, and DNA synthesis, and production of hydroxyl radicals, 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 results in bacterial death. We excluded other potential bactericidal mechanisms (inhibition of extracellular peptidoglycan synthesis, hydrolysis of peptidoglycan, and membrane permeabilization). Thus we reveal a novel mechanism of bacterial killing by innate immunity proteins that bind to cell wall or outer membrane and exploit bacterial stress defense response to kill bacteria.

Seminal regulatory controls of microbial arsenite [As(III)] oxidation are described in this study. Transposon mutagenesis of Agrobacterium tumefaciens identified genes essential for As(III) oxidation, including those coding for a two-component signal transduction pair. The transposon interrupted a response regulator gene (referred to as aoxR), which encodes an ntrC-like protein and is immediately downstream of a gene (aoxS) encoding a protein with primary structural features found in sensor histidine kinases. The structural genes for As(III) oxidase (aoxAB), a c-type cytochrome (cytc 2 ), and molybdopterin biosynthesis (chlE) were downstream of aoxR. The mutant could not be complemented by aoxSR in trans but was complemented by a clone containing aoxS-aoxR-aoxA-aoxB-cytc 2 and consistent with reverse transcriptase (RT) PCR experiments, which demonstrated these genes are cotranscribed as an operon. Expression of aoxAB was monitored by RT-PCR and found to be up-regulated by the addition of As(III) to cell cultures. Expression of aoxAB was also controlled in a fashion consistent with quorum sensing in that (i) expression of aoxAB was absent in As(III)-unexposed early-log-phase cells but was observed in As(III)-unexposed, late-log-phase cells and (ii) treating As(III)-unexposed, early-log-phase cells with ethyl acetate extracts of As(III)-unexposed, late-log-phase culture supernatants also resulted in aoxAB induction. Under inducing conditions, aoxS expression was readily observed in the wild-type strain but significantly reduced in the mutant, indicating that AoxR is autoregulatory and at least partially controls the expression of the aox operon. In summary, regulation of A. tumefaciens As(III) oxidation is complex, apparently being controlled by As(III) exposure, a two-component signal transduction system, and quorum sensing.Arsenic (As) is a known carcinogen, occurring in the environment primarily as the oxyanion arsenate (H 2 AsO 4 Ϫ , HAsO 4 2Ϫ ) [As(V)] and arsenite (H 3 AsO 3 0 ) [As(III)]. As(III) is more toxic and generally the more mobile species (reviewed in reference 6), and hence, there is significant interest in understanding the factors that control As speciation in the environment. Both abiotic and biotic factors are involved; however, it is now generally viewed that microbial As redox transformations are important, if not principal, drivers controlling As speciation in soils, sediments, and natural waters (6, 20).Microbial As(III) oxidation and As(V) reduction activities are cellular strategies for either detoxification or for generating energy. A fairly detailed model explaining the genetics, regulation, and function of detoxification-based As(V) reduction is in place (32), but beyond that little is known. Genes encoding respiratory As(V) reductases have been cloned and enzyme characterizations are under way (recently reviewed in reference 32). Likewise, genes encoding As(III) oxidase have also recently been cloned and characterized (17,29), and extensive enzyme characterization has been achieved (...

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.