Paul E. Carlson

Antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to Clostridium difficile infection

Antibiotics can have significant and long lasting effects on the gastrointestinal tract microbiota, reducing colonization resistance against pathogens including Clostridium difficile. Here we show that antibiotic treatment induces substantial changes in the gut microbial community and in the metabolome of mice susceptible to C. difficile infection. Levels of secondary bile acids, glucose, free fatty acids, and dipeptides decrease, whereas those of primary bile acids and sugar alcohols increase, reflecting the modified metabolic activity of the altered gut microbiome. In vitro and ex vivo analyses demonstrate that C. difficile can exploit specific metabolites that become more abundant in the mouse gut after antibiotics, including primary bile acid taurocholate for germination, and carbon sources mannitol, fructose, sorbitol, raffinose and stachyose for growth. Our results indicate that antibiotic-mediated alteration of the gut microbiome converts the global metabolic profile to one that favors C. difficile germination and growth.

Cefoperazone-treated mice as an experimental platform to assess differential virulence of Clostridium difficile strains.

The toxin-producing bacterium C. difficile is the leading cause of antibiotic-associated colitis, with an estimated 500,000 cases C. difficile infection (CDI) each year in the US with a cost approaching 3 billion dollars. Despite the significance of CDI, the pathogenesis of this infection is still being defined. The recent development of tractable murine models of CDI will help define the determinants of C. difficile pathogenesis in vivo. To determine if cefoperazone-treated mice could be utilized to reveal differential pathogenicity of C. difficile strains, 5–8 week old C57BL/6 mice were pretreated with a 10 d course of cefoperazone administered in the drinking water. Following a 2-d recovery period without antibiotics, the animals were orally challenged with C. difficile strains chosen to represent the potential range of virulence of this organism from rapidly fatal to nonpathogenic. Animals were monitored for loss of weight and clinical signs of colitis. At the time of harvest, C. difficile strains were isolated from cecal contents and the severity of colitis was determined by histopathologic examination of the cecum and colon. Cefoperazone treated mice challenged with C. difficile strains VPI 10463 and BI1 exhibited signs of severe colitis while infection with 630 and F200 was subclinical. This increased clinical severity was correlated with more severe histopathology with significantly more edema, inflammation and epithelial damage encountered in the colons of animals infected with VPI 10463 and BI1. Disease severity also correlated with levels of C. difficile cytotoxic activity in intestinal tissues and elevated blood neutrophil counts. Cefoperazone treated mice represent a useful model of C. difficile infection that will help us better understand the pathogenesis and virulence of this re-emerging pathogen.

Transcriptional Profiling of Bacillus anthracis Sterne (34F2) during Iron Starvation

Lack of available iron is one of many environmental challenges that a bacterium encounters during infection and adaptation to iron starvation is important for the pathogen to efficiently replicate within the host. Here we define the transcriptional response of B. anthracis Sterne (34F2) to iron depleted conditions. Genome-wide transcript analysis showed that B. anthracis undergoes considerable changes in gene expression during growth in iron-depleted media, including the regulation of known and candidate virulence factors. Two genes encoding putative internalin proteins were chosen for further study. Deletion of either gene (GBAA0552 or GBAA1340) resulted in attenuation in a murine model of infection. This attenuation was amplified in a double mutant strain. These data define the transcriptional changes induced during growth in low iron conditions and illustrate the potential of this dataset in the identification of putative virulence determinants for future study.

The relationship between phenotype, ribotype, and clinical disease in human Clostridium difficile isolates.

Since 2000, Clostridium difficile isolates of ribotype 027 have been linked to outbreaks in North America and Europe and also an increased rate of colectomy and death among infected individuals. It has been proposed that enhanced sporulation and toxin production were associated with this apparent increase in virulence of 027 isolates. Since only a limited number of isolates have been examined, the relationship of these phenotypes to a specific ribotype, and as well as to clinical disease severity, remains controversial. 106 recent clinical isolates from the University of Michigan Health System were characterized for the ability to sporulate, produce viable spores, grow in rich media, and produce toxins in vitro. Significant variation was observed between isolates for each of these phenotypes. Isolates of ribotype 027 produced higher levels of toxin and exhibited slower growth compared to other ribotypes. Importantly, increased spore production did appear to be relevant to severe C. difficile infection, as determined by available clinical meta-data. These data provide the first significant difference between isolates from severe vs. less severe disease based on an in vitro C. difficile phenotype and suggest that clinical outcome is better predicted by bacterial attributes other than ribotype.

Genetic analysis of petrobactin transport in Bacillus anthracis

Iron acquisition mechanisms play an important role in the pathogenesis of many infectious microbes. In Bacillus anthracis, the siderophore petrobactin is required for both growth in iron‐depleted conditions and for full virulence of the bacterium. Here we demonstrate the roles of two putative petrobactin binding proteins FatB and FpuA (encoded by GBAA5330 and GBAA4766 respectively) in B. anthracis iron acquisition and pathogenesis. Markerless deletion mutants were created using allelic exchange. The ΔfatB strain was capable of wild‐type levels of growth in iron‐depleted conditions, indicating that FatB does not play an essential role in petrobactin uptake. In contrast, ΔfpuA bacteria exhibited a significant decrease in growth under low‐iron conditions when compared with wild‐type bacteria. This mutant could not be rescued by the addition of exogenous purified petrobactin. Further examination of this strain demonstrated increased levels of petrobactin accumulation in the culture supernatants, suggesting no defect in siderophore synthesis or export but, instead, an inability of ΔfpuA to import this siderophore. ΔfpuA spores were also significantly attenuated in a murine model of inhalational anthrax. These results provide the first genetic evidence demonstrating the role of FpuA in petrobactin uptake.

Membrane Topology of the Bacillus anthracis GerH Germinant Receptor Proteins

Bacillus anthracis spores are the etiologic agent of anthrax. Nutrient germinant receptors (nGRs) packaged within the inner membrane of the spore sense the presence of specific stimuli in the environment and trigger the process of germination, quickly returning the bacterium to the metabolically active, vegetative bacillus. This ability to sense the host environment and initiate germination is a required step in the infectious cycle. The nGRs are comprised of three subunits: the A-, B-, and C-type proteins. To date there are limited structural data for the A- and B-type nGR subunits. Here the transmembrane topologies of the B. anthracis GerH(A), GerH(B), and GerH(C) proteins are presented. C-terminal green fluorescent protein (GFP) fusions to various lengths of the GerH proteins were overexpressed in vegetative bacteria, and the subcellular locations of these GFP fusion sites were analyzed by flow cytometry and protease sensitivity. GFP fusion to full-length GerH(C) confirmed that the C terminus of this protein is extracellular, as predicted. GerH(A) and GerH(B) were both predicted to be integral membrane proteins by topology modeling. Analysis of C-terminal GFP fusions to full-length GerH(B) and nine truncated GerH(B) proteins supports either an 8- or 10-transmembrane-domain topology. For GerH(A), C-terminal GFP fusions to full-length GerH(A) and six truncated GerH(A) proteins were consistent with a four-transmembrane-domain topology. Understanding the membrane topology of these proteins is an important step in determining potential ligand binding and protein-protein interaction domains, as well as providing new information for interpreting previous genetic work.

Intestinal calcium and bile salts facilitate germination of Clostridium difficile spores

Clostridium difficile (C. difficile) is an anaerobic gram-positive pathogen that is the leading cause of nosocomial bacterial infection globally. C. difficile infection (CDI) typically occurs after ingestion of infectious spores by a patient that has been treated with broad-spectrum antibiotics. While CDI is a toxin-mediated disease, transmission and pathogenesis are dependent on the ability to produce viable spores. These spores must become metabolically active (germinate) in order to cause disease. C. difficile spore germination occurs when spores encounter bile salts and other co-germinants within the small intestine, however, the germination signaling cascade is unclear. Here we describe a signaling role for Ca2+ during C. difficile spore germination and provide direct evidence that intestinal Ca2+ coordinates with bile salts to stimulate germination. Endogenous Ca2+ (released from within the spore) and a putative AAA+ ATPase, encoded by Cd630_32980, are both essential for taurocholate-glycine induced germination in the absence of exogenous Ca2+. However, environmental Ca2+ replaces glycine as a co-germinant and circumvents the need for endogenous Ca2+ fluxes. Cd630_32980 is dispensable for colonization in a murine model of C. difficile infection and ex vivo germination in mouse ileal contents. Calcium-depletion of the ileal contents prevented mutant spore germination and reduced WT spore germination by 90%, indicating that Ca2+ present within the gastrointestinal tract plays a critical role in C. difficile germination, colonization, and pathogenesis. These data provide a biological mechanism that may explain why individuals with inefficient intestinal calcium absorption (e.g., vitamin D deficiency, proton pump inhibitor use) are more prone to CDI and suggest that modulating free intestinal calcium is a potential strategy to curb the incidence of CDI.

Interleukin‐22 and CD160 play additive roles in the host mucosal response to Clostridium difficile infection in mice

Our previous work has shown the significant up‐regulation of Il22 and increased phosphorylation of signal transducer and activator of transcription 3 (STAT3) as part of the mucosal inflammatory response to Clostridium difficile infection in mice. Others have shown that phosphorylation of STAT3 at mucosal surfaces includes interleukin‐22 (IL‐22) and CD160‐mediated components. The current study sought to determine the potential role(s) of IL‐22 and/or CD160 in the mucosal response to C. difficile infection. Clostridium difficile‐infected mice treated with anti‐IL‐22, anti‐CD160 or a combination of the two showed significantly reduced STAT3 phosphorylation in comparison to C. difficile‐infected mice that had not received either antibody. In addition, C. difficile‐infected mice treated with anti‐IL‐22/CD160 induced a smaller set of genes, and at significantly lower levels than the untreated C. difficile‐infected mice. The affected genes included pro‐inflammatory chemokines and cytokines, and anti‐microbial peptides. Furthermore, histopathological and flow cytometric assessments both showed a significantly reduced influx of neutrophils in C. difficile‐infected mice treated with anti‐IL‐22/CD160. These data demonstrate that IL‐22 and CD160 are together responsible for a significant fraction of the colonic STAT3 phosphorylation in C. difficile infection. They also underscore the additive effects of IL‐22 and CD160 in mediating both the pro‐inflammatory and pro‐survival aspects of the host mucosal response in this infection.

Multiple ABC transporters are involved in the acquisition of petrobactin in Bacillus anthracis

In Bacillus anthracis the siderophore petrobactin is vital for iron acquisition and virulence. The petrobactin‐binding receptor FpuA is required for these processes. Here additional components of petrobactin reacquisition are described. To identify these proteins, mutants of candidate permease and ATPase genes were generated allowing for characterization of multiple petrobactin ATP‐binding cassette (ABC)‐import systems. Either of two distinct permeases, FpuB or FatCD, is required for iron acquisition and play redundant roles in petrobactin transport. A mutant strain lacking both permeases, ΔfpuBΔfatCD, was incapable of using petrobactin as an iron source and exhibited attenuated virulence in a murine model of inhalational anthrax infection. ATPase mutants were generated in either of the permease mutant backgrounds to identify the ATPase(s) interacting with each individual permease channel. Mutants lacking the FpuB permease and FatE ATPase (ΔfpuBΔfatE) and a mutant lacking the distinct ATPases FpuC and FpuD generated in the ΔfatCD background (ΔfatCDΔfpuCΔfpuD) displayed phenotypic characteristics of a mutant deficient in petrobactin import. A mutant lacking all three of the identified ATPases (ΔfatEΔfpuCΔfpuD) exhibited the same growth defect in iron‐depleted conditions. Taken together, these results provide the first description of the permease and ATPase proteins required for the import of petrobactin in B. anthracis.

Flying under the radar

The dramatic, rapid growth of Bacillus anthracis that occurs during systemic anthrax implies a crucial requirement for the efficient acquisition of iron. While recent advances in our understanding of B. anthracis iron acquisition systems indicate the use of strategies similar to other pathogens, this review focuses on unique features of the major siderophore system, petrobactin. Ways that petrobactin differs from other siderophores include: A. unique ferric iron binding moieties that allow petrobactin to evade host immune proteins; B. a biosynthetic operon that encodes enzymes from both major siderophore biosynthesis classes; C. redundancy in membrane transport systems for acquisition of Fe‐petrobactin holo‐complexes; and, D. regulation that appears to be controlled predominately by sensing the host‐like environmental signals of temperature, CO2 levels and oxidative stress, as opposed to canonical sensing of intracellular iron levels. We argue that these differences contribute in meaningful ways to B. anthracis pathogenesis. This review will also outline current major gaps in our understanding of the petrobactin iron acquisition system, some projected means for exploiting current knowledge, and potential future research directions.