A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families known to be produced by the microbiome have a profound impact on the balance between health and disease 1-9. Considering the diversity of the human microbiome, numbering over 40,000 operational taxonomic units 10 , the impact of the microbiome on the chemistry of an entire animal remains underexplored. In this study, mass spectrometry informatics and data visualization approaches 11-13 were used to provide an assessment of the impacts of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free (GF) and specific pathogen Reprints and/or permissions can be provided by R. Quinn or P.C. Dorrestein.

Summary Pathogenic Yersinia, including Y. pestis, the agent of plague in humans, and Y. pseudotuberculosis, the related enteric pathogen, deliver virulence effectors into host cells via a prototypical type III secretion system to promote pathogenesis. These effectors, termed Yersinia outer proteins (Yops), modulate multiple host signaling responses. Studies in Y. pestis and Y. pseudotuberculosis have shown that YopM suppresses infection-induced inflammasome activation, however the underlying molecular mechanism is largely unknown. Here we show that YopM specifically restricts the pyrin inflammasome, which is triggered by the RhoA-inactivating enzymatic activities of YopE and YopT, in Y. pseudotuberculosis-infected macrophages. The attenuation of a yopM mutant is fully reversed in pyrin knock-out mice, demonstrating that YopM inhibits pyrin to promote virulence. Mechanistically, YopM recruits and activates the host kinases PRK1 and PRK2 to negatively regulate pyrin by phosphorylation. These results show how a virulence factor can hijack host kinases to inhibit effector-triggered pyrin inflammasome activation.

Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by homozygous or compound heterozygous gain-of-function mutations in MEFV , encoding pyrin, an inflammasome protein. Heterozygous carrier frequencies for multiple MEFV mutations are high in several Mediterranean populations, suggesting that they confer selective advantage. Among 2,313 Turks, we found extended haplotype homozygosity flanking FMF-associated mutations, indicating evolutionarily recent positive selection of FMF-associated mutations. Two pathogenic pyrin variants independently arose >1,800 years ago. Mutant pyrin interacts less avidly with Yersinia pestis virulence factor YopM than wild type human pyrin, thereby attenuating YopM-induced IL-1β suppression. Relative to healthy controls, leukocytes from FMF patients harboring homozygous or compound heterozygous mutations and from asymptomatic heterozygous carriers released heightened IL-1β specifically in response to Y. pestis . Y. pestis -infected Mefv M680I/M680I FMF knock-in mice exhibited IL-1-dependent increased survival relative to wild-type knock-in mice. Thus, FMF mutations that were positively selected in Mediterranean populations confer heightened resistance to Y. pestis .

We have identified two novel proteins that colocalize with the subpellicular microtubules in the protozoan parasite Toxoplasma gondii and named these proteins SPM1 and SPM2. These proteins have basic isoelectric points and both have homologs in other apicomplexan parasites. SPM1 contains six tandem copies of a 32-amino-acid repeat, whereas SPM2 lacks defined protein signatures. Alignment of Toxoplasma SPM2 with apparent Plasmodium SPM2 homologs indicates that the greatest degree of conservation lies in the carboxy-terminal half of the protein. Analysis of Plasmodium homologs of SPM1 indicates that while the central 32-amino-acid repeats have expanded to different degrees (7, 8, 9, 12, or 13 repeats), the amino-and carboxy-terminal regions remain conserved. In contrast, although the Cryptosporidium SPM1 homolog has a conserved carboxy tail, the five repeats are considerably diverged, and it has a smaller amino-terminal domain. SPM1 is localized along the full length of the subpellicular microtubules but does not associate with the conoid or spindle microtubules. SPM2 has a restricted localization along the middle region of the subpellicular microtubules. Domain deletion analysis indicates that four or more copies of the SPM1 repeat are required for localization to microtubules, and the amino-terminal 63 residues of SPM2 are required for localization to the subpellicular microtubules. Gene deletion studies indicate that neither SPM1 nor SPM2 is essential for tachyzoite viability. However, loss of SPM1 decreases overall parasite fitness and eliminates the stability of subpellicular microtubules to detergent extraction.

YopM is a leucine-rich repeat (LRR)-containing effector in several Yersinia species, including Yersinia pestis and Y. pseudotuberculosis. Different Yersinia strains encode distinct YopM isoforms with variable numbers of LRRs but conserved C-terminal tails. A 15-LRR isoform in Y. pseudotuberculosis YPIII was recently shown to bind and inhibit caspase-1 via a YLTD motif in LRR 10, and attenuation of YopM− YPIII was reversed in mice lacking caspase-1, indicating that caspase-1 inhibition is a major virulence function of YopMYPIII. To determine if other YopM proteins inhibit caspase-1, we utilized Y. pseudotuberculosis strains natively expressing a 21-LRR isoform lacking the YLTD motif (YopM32777) or ectopically expressing a Y. pestis 15-LRR version with a functional (YopMKIM) or inactivated (YopMKIM D271A) YLTD motif. Results of mouse and macrophage infections with these strains showed that YopM32777, YopMKIM, and YopMKIM D271A inhibit caspase-1 activation, indicating that the YLTD motif is dispensable for this activity. Analysis of YopMKIM deletion variants revealed that LRRs 6 to 15 and the C-terminal tail are required to inhibit caspase-1 activation. YopM32777, YopMKIM, and YopMKIM deletion variants were purified, and binding partners in macrophage lysates were identified. Caspase-1 bound to YopMKIM but not YopM32777. Additionally, YopMKIM bound IQGAP1 and the use of Iqgap1−/− macrophages revealed that this scaffolding protein is important for caspase-1 activation upon infection with YopM− Y. pseudotuberculosis. Thus, while multiple YopM isoforms inhibit caspase-1 activation, their variable LRR domains bind different host proteins to perform this function and the LRRs of YopMKIM target IQGAP1, a novel regulator of caspase-1, in macrophages.

The gastrointestinal tract is a complex environment in which the host immune system interacts with a diverse array of microorganisms, both symbiotic and pathogenic. As such, mobilizing a rapid and appropriate antimicrobial response depending on the nature of each stimulus is crucial for maintaining the balance between homeostasis and inflammation in the gut. Here we focus on the mechanisms by which intestinal antimicrobial peptides regulate microbial communities during dysbiosis and infection. We also discuss classes of bacterial peptides that contribute to reducing enteric pathogen outgrowth. This review aims to provide a comprehensive overview on the interplay of diverse antimicrobial responses with enteric pathogens and the gut microbiota.

Being the first line of defense against invading pathogens, the innate immune system has evolved to respond to general patterns of infection, termed pathogen-associated molecular patterns (PAMPs), via pattern recognition receptors (PRRs) (1, 2). Tolllike receptors (TLRs), acting as PRRs, detect distinct PAMPs from invading bacteria, such as flagellin (TLR5) and lipopolysaccharide (LPS; TLR4). Upon PAMP recognition, TLRs activate mitogenactivated protein kinase (MAPK) and nuclear factor-B (NF-B) pathways, which direct production of host-protective factors such as proinflammatory cytokines (3, 4). Bacterial pathogens produce virulence factors that inhibit PRR-directed innate immune responses in order to promote infection (5, 6). For example, numerous Gram-negative bacterial pathogens encode type III secretion systems (T3SSs) that are used to counteract host innate immune responses (7). During bacterium-host cell contact, T3SSs are activated and translocate effectors across the plasma membrane and into the eukaryotic cytosol. Translocated effectors inhibit innate immune responses and promote pathogenesis (7,8). In order to counteract infection by virulent pathogens, host cells can sense perturbations caused by T3SSs and/or effectors and produce heightened innate immune responses (9, 10).Disruptions induced by T3SSs and/or effectors in macrophages infected with bacterial pathogens commonly result in the activation of caspase-1 (11-14). Active caspase-1 promotes maturation and secretion of cytokines such as interleukin-1␤ (IL-1␤) and IL-18, which are produced as inactive precursors. Caspase-1 is also produced as a proenzyme, and its activation typically occurs in an inflammasome, a multioligomeric complex that serves as a molecular platform for the recruitment and auto-proteolytic cleavage of procaspase-1 (11-13, 15). Active caspase-1 also induces lysosome exocytosis and a form of inflammatory cell death, termed pyroptosis, which is characterized by osmotic swelling of the macrophage and subsequent rupture of the plasma membrane, resulting in the release of proinflammatory molecules (16). Several inflammasomes have been identified, and all but one (that formed by AIM2) contain proteins that are part of the nucleotidebinding domain leucine-rich repeat (NLR) family. These NLRs serve as cytosolic PRRs and are activated upon recognition of cytosolic PAMPs or danger signals (2,11,15). For some NLRs, such as NLRP3, the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) mediates inflammasome assembly by interacting with capase-1 and the NLR (11,13,15). Complexes of NLRs and ASC form large structures in macrophages that can be detected as foci by microscopic techniques (17)(18)(19). In cases where inflammasome assembly is

The human pathogenic Yersinia species cause diseases that represent a significant source of morbidity and mortality. Despite this, specific mechanisms underlying Yersinia pathogenesis and protective host responses remain poorly understood. Recent studies have shown that Yersinia disrupt cell death pathways, perturb inflammatory processes and exploit immune cells to promote disease. The ensuing host responses following Yersinia infection include coordination of innate and adaptive immune responses in an attempt to control bacterial replication. Here, we highlight current advances in our understanding of the interactions between the pathogenic yersiniae and host cells, as well as the protective host responses mobilized to counteract these pathogens. Together, these studies enhance our understanding of Yersinia pathogenesis and highlight the ongoing battle between host and microbe.