Doctor of Philosophy, University of Wisconsin Madison (2014)
Bachelor of Science, University of Pittsburgh (2009)
Justin Sonnenburg, Postdoctoral Faculty Sponsor
Sepsis is a deleterious immune response to infection that leads to organ failure and is the 11th most common cause of death worldwide. Despite plaguing humanity for thousands of years, the host factors that regulate this immunological response and subsequent sepsis severity and outcome are not fully understood. Here we describe how the Western diet (WD), a diet high in fat and sucrose and low in fiber, found rampant in industrialized countries, leads to worse disease and poorer outcomes in an LPS-driven sepsis model in WD-fed mice compared with mice fed standard fiber-rich chow (SC). We find that WD-fed mice have higher baseline inflammation (metaflammation) and signs of sepsis-associated immunoparalysis compared with SC-fed mice. WD mice also have an increased frequency of neutrophils, some with an "aged" phenotype, in the blood during sepsis compared with SC mice. Importantly, we found that the WD-dependent increase in sepsis severity and higher mortality is independent of the microbiome, suggesting that the diet may be directly regulating the innate immune system through an unknown mechanism. Strikingly, we could predict LPS-driven sepsis outcome by tracking specific WD-dependent disease factors (e.g., hypothermia and frequency of neutrophils in the blood) during disease progression and recovery. We conclude that the WD is reprogramming the basal immune status and acute response to LPS-driven sepsis and that this correlates with alternative disease paths that lead to more severe disease and poorer outcomes.
View details for PubMedID 30808756
Clostridium difficile is an opportunistic diarrhoeal pathogen, and C.?difficile infection (CDI) represents a major health care concern, causing an estimated 15,000 deaths per year in the United States alone 1 . Several enteric pathogens, including C.?difficile, leverage inflammation and the accompanying microbial dysbiosis to thrive in the distal gut 2 . Although diet is among the most powerful available tools for affecting the health of humans and their relationship with their microbiota, investigation into the effects of diet on CDI has been limited. Here, we show in mice that the consumption of microbiota-accessible carbohydrates (MACs) found in dietary plant polysaccharides has a significant effect on CDI. Specifically, using a model of antibiotic-induced CDI that typically resolves within 12?days of infection, we demonstrate that MAC-deficient diets perpetuate CDI. We show that C.?difficile burdens are suppressed through the addition of either a diet containing a complex mixture of MACs or a simplified diet containing inulin as the sole MAC source. We show that switches between these dietary conditions are coincident with changes to microbiota membership, its metabolic output and C.?difficile-mediated inflammation. Together, our data demonstrate the outgrowth of MAC-utilizing taxa and the associated end products of MAC metabolism, namely, the short-chain fatty acids acetate, propionate and butyrate, are associated with decreased C.?difficile fitness despite increased C.?difficile toxin expression in the gut. Our findings, when placed into the context of the known fibre deficiencies of a human Western diet, provide rationale for pursuing MAC-centric dietary strategies as an alternate line of investigation for mitigating CDI.
View details for PubMedID 29686297
The human gut microbiota produces dozens of metabolites that accumulate in the bloodstream, where they can have systemic effects on the host. Although these small molecules commonly reach concentrations similar to those achieved by pharmaceutical agents, remarkably little is known about the microbial metabolic pathways that produce them. Here we use a combination of genetics and metabolic profiling to characterize a pathway from the gut symbiont Clostridium sporogenes that generates aromatic amino acid metabolites. Our results reveal that this pathway produces twelve compounds, nine of which are known to accumulate in host serum. All three aromatic amino acids (tryptophan, phenylalanine and tyrosine) serve as substrates for the pathway, and it involves branching and alternative reductases for specific intermediates. By genetically manipulating C. sporogenes, we modulate serum levels of these metabolites in gnotobiotic mice, and show that in turn this affects intestinal permeability and systemic immunity. This work has the potential to provide the basis of a systematic effort to engineer the molecular output of the gut bacterial community.
View details for PubMedID 29168502
It is widely accepted that Clostridium difficile exploits dysbiosis and leverages inflammation to thrive in the gut environment, where it can asymptomatically colonize humans or cause a toxin-mediated disease ranging in severity from frequent watery diarrhea to pseudomembranous colitis or toxic megacolon. Here, we synthesize recent findings from the gut microbiota and enteric pathogenesis fields to inform the next steps toward a better understanding of C. difficile infection (CDI). In this review, we present a model in which the lifestyle of C. difficile is dictated by the metabolic state of the distal gut ecosystem. Contributions by C. difficile (specifically the production and action of the large glycosylating toxins TcdA and TcdB), the microbiota, and the host dictate whether the gut environment is supportive to the pathogen. Mechanistic, metabolic pathway-focused approaches encompassing the roles of all of these players are helping to elucidate the molecular ecology of the distal gut underlying a diseased or healthy ecosystem. A new generation of therapeutic strategies that are more targeted (and palatable) than fecal microbiota transplants or broad-spectrum antibiotics will be fueled by insight into the interspecies (host-microbe and microbe-microbe) interactions that differentiate healthy from pathogen-infested microbiotas.
View details for DOI 10.1016/j.mib.2016.11.006
View details for PubMedID 27997854
Clostridium difficile infection (CDI) is a worldwide health threat that is typically triggered by the use of broad-spectrum antibiotics, which disrupt the natural gut microbiota and allow this Gram-positive anaerobic pathogen to thrive. The increased incidence and severity of disease coupled with decreased response, high recurrence rates, and emergence of multiple antibiotic-resistant strains have created an urgent need for new therapies. We describe pharmacological targeting of the cysteine protease domain (CPD) within the C. difficile major virulence factor toxin B (TcdB). Through a targeted screen with an activity-based probe for this protease domain, we identified a number of potent CPD inhibitors, including one bioactive compound, ebselen, which is currently in human clinical trials for a clinically unrelated indication. This drug showed activity against both major virulence factors, TcdA and TcdB, in biochemical and cell-based studies. Treatment in a mouse model of CDI that closely resembles the human infection confirmed a therapeutic benefit in the form of reduced disease pathology in host tissues that correlated with inhibition of the release of the toxic glucosyltransferase domain (GTD). Our results show that this non-antibiotic drug can modulate the pathology of disease and therefore could potentially be developed as a therapeutic for the treatment of CDI.
View details for DOI 10.1126/scitranslmed.aac9103
View details for Web of Science ID 000365232600002
View details for PubMedID 26400909
The urinary tract environment provides many conditions that deter colonization by microorganisms. D-serine is thought to be one of these stressors and is present at high concentrations in urine. D-serine interferes with L-serine and pantothenate metabolism and is bacteriostatic to many species. Uropathogenic Escherichia coli commonly possess the dsdCXA genetic locus, which allows them to use D-serine as a sole carbon, nitrogen, and energy source. It was previously reported that in the model UPEC strain CFT073, a dsdA mutant outcompetes wild type in the murine model of urinary tract infection. This "hypercolonization" was used to propose a model whereby UPEC strains sense D-serine in the urinary tract and subsequently up-regulate genes necessary for pathogenesis. Here, we show that inactivation of dsdA does not lead to hypercolonization. We suggest that this previously observed effect is due to an unrecognized secondary mutation in rpoS and that some D-serine specific effects described in other studies may be affected by the rpoS status of the strains used. Inactivation of dsdA in the original clinical isolate of CFT073 gives CFT073 ?dsdA a growth defect in human urine and renders it unable to grow on minimal medium containing D-serine as the sole carbon source. However, CFT073 ?dsdA is able to colonize the urinary tracts of CBA/J mice indistinguishably from wild type. These findings indicate that D-serine catabolism, though it may play role(s) during urinary tract infection, does not affect the ability of uropathogenic E. coli to colonize the murine urinary tract.
View details for DOI 10.1371/journal.pone.0138121
View details for PubMedID 26366567
Clostridium difficile is a leading cause of antibiotic-associated diarrhea. The mechanisms underlying C. difficile expansion after microbiota disturbance are just emerging. We assessed the gene expression profile of C. difficile within the intestine of gnotobiotic mice to identify genes regulated in response to either dietary or microbiota compositional changes. In the presence of the gut symbiont Bacteroides thetaiotaomicron, C. difficile induces a pathway that metabolizes the microbiota fermentation end-product succinate to butyrate. The low concentration of succinate present in the microbiota of conventional mice is transiently elevated upon antibiotic treatment or chemically induced intestinal motility disturbance, and C. difficile exploits this succinate spike to expand in the perturbed intestine. A C. difficile mutant compromised in succinate utilization is at a competitive disadvantage during these perturbations. Understanding the metabolic mechanisms involved in microbiota-C. difficile interactions may help to identify approaches for the treatment and prevention of C. difficile-associated diseases.
View details for DOI 10.1016/j.chom.2014.11.003
View details for Web of Science ID 000346211100010
View details for PubMedID 25498344
RpoS (?(S)), the general stress response sigma factor, directs the expression of genes under a variety of stressful conditions. Control of the cellular ?(S) concentration is critical for appropriately scaled ?(S)-dependent gene expression. One way to maintain appropriate levels of ?(S) is to regulate its stability. Indeed, ?(S) degradation is catalyzed by the ClpXP protease and the recognition of ?(S) by ClpXP depends on the adaptor protein RssB. Three anti-adaptors (IraD, IraM, and IraP) exist in Escherichia coli K-12; each interacts with RssB and inhibits RssB activity under different stress conditions, thereby stabilizing ?(S). Unlike K-12, some E. coli isolates, including uropathogenic E. coli strain CFT073, show comparable cellular levels of ?(S) during the logarithmic and stationary growth phases, suggesting that there are differences in the regulation of ?(S) levels among E. coli strains. Here, we describe IraL, an RssB anti-adaptor that stabilizes ?(S) during logarithmic phase growth in CFT073 and other E. coli and Shigella strains. By immunoblot analyses, we show that IraL affects the levels and stability of ?(S) during logarithmic phase growth. By computational and PCR-based analyses, we reveal that iraL is found in many E. coli pathotypes but not in laboratory-adapted strains. Finally, by bacterial two-hybrid and copurification analyses, we demonstrate that IraL interacts with RssB by a mechanism distinct from that used by other characterized anti-adaptors. We introduce a fourth RssB anti-adaptor found in E. coli species and suggest that differences in the regulation of ?(S) levels may contribute to host and niche specificity in pathogenic and nonpathogenic E. coli strains.Bacteria must cope with a variety of environmental conditions in order to survive. RpoS (?(S)), the general stress response sigma factor, directs the expression of many genes under stressful conditions in both pathogenic and nonpathogenic Escherichia coli strains. The regulation of ?(S) levels and activity allows appropriately scaled ?(S)-dependent gene expression. Here, we describe IraL, an RssB anti-adaptor that, unlike previously described anti-adaptors, stabilizes ?(S) during the logarithmic growth phase in the absence of additional stress. We also demonstrate that iraL is found in a large number of E. coli and Shigella isolates. These data suggest that strains containing iraL are able to initiate ?(S)-dependent gene expression under conditions under which strains without iraL cannot. Therefore, IraL-mediated ?(S) stabilization may contribute to host and niche specificity in E. coli.
View details for DOI 10.1128/mBio.01043-14
View details for PubMedID 24865554
Uropathogenic Escherichia coli (UPEC) is the most common causative agent of community-acquired urinary tract infection (UTI). In order to cause UTI, UPEC must endure stresses ranging from nutrient limitation to host immune components. RpoS (?(S)), the general stress response sigma factor, directs gene expression under a variety of inhibitory conditions. Our study of rpoS in UPEC strain CFT073 began after we discovered an rpoS-frameshift mutation in one of our laboratory stocks of "wild-type" CFT073. We demonstrate that an rpoS-deletion mutation in CFT073 leads to a colonization defect during UTI of CBA/J mice at 48 hours postinfection (hpi). There is no difference between the growth rates of CFT073 and CFT073 rpoS in urine. This indicates that rpoS is needed for replication and survival in the host rather than being needed to address limitations imposed by urine nutrients. Consistent with previous observations in E. coli K-12, CFT073 rpoS is more sensitive to oxidative stress than the wild type. We demonstrate that peroxide levels are elevated in voided urine from CFT073-infected mice compared to urine from mock-infected mice, which supports the notion that oxidative stress is generated by the host in response to UPEC. In mice that lack phagocyte oxidase, the enzyme complex expressed by phagocytes that produces superoxide, the competitive defect of CFT073 rpoS in bladder colonization is lost. These results demonstrate that ?(S) is important for UPEC survival under conditions of phagocyte oxidase-generated stress during UTI. Though ?(S) affects the pathogenesis of other bacterial species, this is the first work that directly implicates ?(S) as important for UPEC pathogenesis.UPEC must cope with a variety of stressful conditions in the urinary tract during infection. RpoS (?(S)), the general stress response sigma factor, is known to direct the expression of many genes under a variety of stressful conditions in laboratory-adapted E. coli K-12. Here, we show that ?(S) is needed by the model UPEC strain CFT073 to cope with oxidative stress provided by phagocytes during infection. These findings represent the first report that implicates ?(S) in the fitness of UPEC during infection and support the idea of the need for a better understanding of the effects of this global regulator of gene expression during UTI.
View details for DOI 10.1128/mBio.00023-13
View details for Web of Science ID 000315814300003
View details for PubMedID 23404396
The traditional genetic procedure for random or site-specific mutagenesis in Escherichia coli K-12 involves mutagenesis, isolation of mutants, and transduction of the mutation into a clean genetic background. The transduction step reduces the likelihood of complications due to secondary mutations. Though well established, this protocol is not tenable for many pathogenic E. coli strains, such as uropathogenic strain CFT073, because it is resistant to known K-12 transducing bacteriophages, such as P1. CFT073 mutants generated via a technique such as lambda Red mutagenesis may contain unknown secondary mutations. Here we describe the isolation and characterization of transducing bacteriophages for CFT073. Seventy-seven phage isolates were acquired from effluent water samples collected from a wastewater treatment plant in Madison, WI. The phages were differentiated by a host sensitivity-typing scheme with a panel of E. coli strains from the ECOR collection and clinical uropathogenic isolates. We found 49 unique phage isolates. These were then examined for their ability to transduce antibiotic resistance gene insertions at multiple loci between different mutant strains of CFT073. We identified 4 different phages capable of CFT073 generalized transduction. These phages also plaque on the model uropathogenic E. coli strains 536, UTI89, and NU14. The highest-efficiency transducing phage, ?EB49, was further characterized by DNA sequence analysis, revealing a double-stranded genome 47,180 bp in length and showing similarity to other sequenced phages. When combined with a technique like lambda Red mutagenesis, the newly characterized transducing phages provide a significant development in the genetic tools available for the study of uropathogenic E. coli.
View details for DOI 10.1128/AEM.05307-11
View details for Web of Science ID 000294691400038
View details for PubMedID 21784916
Mycobacteriophages are viruses that infect mycobacterial hosts. Expansion of a collection of sequenced phage genomes to a total of 60-all infecting a common bacterial host-provides further insight into their diversity and evolution. Of the 60 phage genomes, 55 can be grouped into nine clusters according to their nucleotide sequence similarities, 5 of which can be further divided into subclusters; 5 genomes do not cluster with other phages. The sequence diversity between genomes within a cluster varies greatly; for example, the 6 genomes in Cluster D share more than 97.5% average nucleotide similarity with one another. In contrast, similarity between the 2 genomes in Cluster I is barely detectable by diagonal plot analysis. In total, 6858 predicted open-reading frames have been grouped into 1523 phamilies (phams) of related sequences, 46% of which possess only a single member. Only 18.8% of the phams have sequence similarity to non-mycobacteriophage database entries, and fewer than 10% of all phams can be assigned functions based on database searching or synteny. Genome clustering facilitates the identification of genes that are in greatest genetic flux and are more likely to have been exchanged horizontally in relatively recent evolutionary time. Although mycobacteriophage genes exhibit a smaller average size than genes of their host (205 residues compared with 315), phage genes in higher flux average only 100 amino acids, suggesting that the primary units of genetic exchange correspond to single protein domains.
View details for DOI 10.1016/j.jmb.2010.01.011
View details for Web of Science ID 000275785600009
View details for PubMedID 20064525
Bacteriophages are the most abundant forms of life in the biosphere and carry genomes characterized by high genetic diversity and mosaic architectures. The complete sequences of 30 mycobacteriophage genomes show them collectively to encode 101 tRNAs, three tmRNAs, and 3,357 proteins belonging to 1,536 "phamilies" of related sequences, and a statistical analysis predicts that these represent approximately 50% of the total number of phamilies in the mycobacteriophage population. These phamilies contain 2.19 proteins on average; more than half (774) of them contain just a single protein sequence. Only six phamilies have representatives in more than half of the 30 genomes, and only three-encoding tape-measure proteins, lysins, and minor tail proteins-are present in all 30 phages, although these phamilies are themselves highly modular, such that no single amino acid sequence element is present in all 30 mycobacteriophage genomes. Of the 1,536 phamilies, only 230 (15%) have amino acid sequence similarity to previously reported proteins, reflecting the enormous genetic diversity of the entire phage population. The abundance and diversity of phages, the simplicity of phage isolation, and the relatively small size of phage genomes support bacteriophage isolation and comparative genomic analysis as a highly suitable platform for discovery-based education.
View details for DOI 10.1371/journal.pgen.0020092
View details for Web of Science ID 000239494700007
View details for PubMedID 16789831