Iron is a transition metal utilized by nearly all forms of life for essential cellular processes, such as DNA synthesis and cellular respiration. During infection by bacterial pathogens, the host utilizes various strategies to sequester iron in a process termed, nutritional immunity. To circumvent these defenses, Gram-negative pathogens have evolved numerous mechanisms to obtain iron from heme. In this review we outline the systems that exist in several Gram-negative pathogens that are associated with heme transport and utilization, beginning with hemolysis and concluding with heme degradation. In addition, Gram-negative pathogens must also closely regulate the intracellular concentrations of iron and heme, since high levels of iron can lead to the generation of toxic reactive oxygen species. As such, we also provide several examples of regulatory pathways that control heme utilization, showing that co-regulation with other cellular processes is complex and often not completely understood.
is a leading cause of bacterially derived gastroenteritis worldwide. is most commonly acquired through the consumption of undercooked poultry meat or through drinking contaminated water. Following ingestion, adheres to the intestinal epithelium and mucus layer, causing toxin-mediated inflammation and inhibition of fluid reabsorption. Currently, the human response to infection is relatively unknown, and animal hosts that model these responses are rare. As such, we examined patient fecal samples for the accumulation of the neutrophil protein calgranulin C during infection with In response to infection, calgranulin C was significantly increased in the feces of humans. To determine whether calgranulin C accumulation occurs in an animal model, we examined disease in ferrets. Ferrets were effectively infected by, with peak fecal loads observed at day 3 postinfection and full resolution by day 12. Serum levels of interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-α) significantly increased in response to infection, which resulted in leukocyte trafficking to the colon. As a result, calgranulin C increased in the feces of ferrets at the time when loads decreased. Further, the addition of purified calgranulin C to cultures was found to inhibit growth in a zinc-dependent manner. These results suggest that upon infection with , leukocytes trafficked to the intestine release calgranulin C as a mechanism for inhibiting growth.
As a leading cause of bacterial-derived gastroenteritis worldwide, Campylobacter jejuni has a significant impact on human health in both the developed and developing worlds. Despite its prevalence as a human pathogen, the source of these infections remains poorly understood due to the mutation frequency of the organism and past limitations of whole genome analysis. Recent advances in both whole genome sequencing and computational methods have allowed for the high-resolution analysis of intraspecies diversity, leading multiple groups to postulate that these approaches may be used to identify the sources of Campylobacter jejuni infection. To address this hypothesis, our group conducted a regionally and temporally restricted sampling of agricultural and environmental Campylobacter sources and compared isolated C. jejuni genomes to those that caused human infections in the same region during the same time period. Through a network analysis comparing genomes from various sources, we found that human C. jejuni isolates clustered with those isolated from cattle and chickens, indicating these as potential sources of human infection in the region.
A previously identified transcriptional regulator in C. jejuni, termed HeuR, was found to positively regulate heme utilization. Additionally, transcriptomic work demonstrated the putative operons, CJJ81176_1390-1394 and CJJ81176_1214-1217, were upregulated in a HeuR mutant, suggesting HeuR negatively regulates expression of these genes. Because genes within these clusters include a cystathionine β-lyase (metC) and a methionine synthase (metE), it appeared HeuR negatively regulates C. jejuni methionine biosynthesis. To address this, we confirmed mutation of HeuR reproducibly results in metC overexpression under nutrient-replete conditions, but did not affect expression of metE, while metC expression in the wild-type increased to heuR mutant levels during iron-limitation. We subsequently determined that both gene clusters are operonic and demonstrated the direct interaction of HeuR with the predicted promoter regions of these operons. Using DNase-footprinting assays, we were able to show that HeuR specifically binds within the predicted -35 region of the CJJ81176_1390-1394 operon. As predicted based on transcriptional results, the HeuR mutant was able to grow and remain viable in a defined media with and without methionine, but we identified significant impacts on growth and viability in metC and metE mutants. Additionally, we observed decreased adherence, invasion, and persistence of metC and metE mutants when incubated with human colonocytes, while the heuR mutant exhibited increased invasion. Taken together, these results suggest that HeuR regulates methionine biosynthesis in an iron-responsive manner and that the ability to produce methionine is an important factor for adhering to and invading the gastrointestinal tract of a susceptible host. Importance As the leading cause of bacterial-derived gastroenteritis worldwide, Campylobacter jejuni has a significant impact on human health. Investigating colonization factors that allow C. jejuni to successfully infect a host furthers our understanding of genes and regulatory elements necessary for virulence. In this study, we have begun to characterize the role of the transcriptional regulatory protein, HeuR, on methionine biosynthesis in C. jejuni. When the ability to synthesize methionine is impaired, detrimental impacts on growth and viability are observed during growth in limited media lacking methionine and/or iron. Additionally, mutations in the methionine biosynthetic pathway result in decreased adhesion, invasion, and intracellular survival of C. jejuni when incubated with human colonocytes, indicating the importance of regulating methionine biosynthesis.
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