Growth of Salmonella enterica Serovars Typhimurium and Enteritidis in Iron-Poor Media and in Meat: Role of Catecholate and Hydroxamate Siderophore Transporters.

Enteritidis and Typhimurium are among the top Salmonella enterica serovars implicated in human salmonellosis worldwide. This study examined the individual and combined roles of catecholate-iron and hydroxamate-iron transporters in the survival in meat of Salmonella Enteritidis and Typhimurium. Catecholate-iron-III (Fe3+) and hydroxamate-Fe3+ transporter genes fepA, iroN, and fhuACDB were deleted in isolates of these serovars to generate single, double, and triple mutants. Growth rate in high- and low-iron media was compared among mutants, complements, and their wild-type parents. Susceptibility to 14 antibiotics, the ability to produce and utilize siderophores, and survival on cooked chicken breast were evaluated. In iron-poor liquid media, differences were observed between the growth characteristics of mutant Salmonella Enteritidis and Typhimurium. The double Δ iroNΔ fepA and the triple Δ fhuΔ iroNΔ fepA mutants of Salmonella Enteritidis exhibited prolonged lag phases (λ = 9.72 and 9.53 h) and a slow growth rate (µmax = 0.35 and 0.25 h-1) similar to that of its Δ tonB mutant (λ = 10.12 h and µmax = 0.30 h-1). In Salmonella Typhimurium, double Δ iroNΔ fepA and triple Δ fhuΔ iroNΔ fepA mutations induced a similar growth pattern as its Δ tonB mutant. Double deletions of fepA and iroN reduced the siderophore production and the use of enterobactin as an iron source. In the Δ iroNΔ fepA mutant, but not in Δ fhuΔ iroNΔ fepA, the ferrichrome or deferrioxamine promoted growth for both serovars, confirming the specific role of the FhuACDB system in the uptake and transport of hydroxamate Fe3+. Survival of the mutants was also evaluated in a meat assay, and no difference in survival was observed among the mutants compared with wild type. This study showed differences between serovars in the importance of catecholate-iron and hydroxamate-iron uptake on Salmonella growth in iron-restricted media. Data also confirmed that both Salmonella Enteritidis and Typhimurium are well equipped to survive on cooked chicken meat, offering a rich iron condition.

Interplay between genetic regulation of phosphate homeostasis and bacterial virulence.

Bacterial pathogens, including those of humans, animals, and plants, encounter phosphate (Pi)-limiting or Pi-rich environments in the host, depending on the site of infection. The environmental Pi-concentration results in modulation of expression of the Pho regulon that allows bacteria to regulate phosphate assimilation pathways accordingly. In many cases, modulation of Pho regulon expression also results in concomitant changes in virulence phenotypes. Under Pi-limiting conditions, bacteria use the transcriptional-response regulator PhoB to translate the Pi starvation signal sensed by the bacterium into gene activation or repression. This regulator is employed not only for the maintenance of bacterial Pi homeostasis but also to differentially regulate virulence. The Pho regulon is therefore not only a regulatory circuit of phosphate homeostasis but also plays an important adaptive role in stress response and bacterial virulence. Here we focus on recent findings regarding the mechanisms of gene regulation that underlie the virulence responses to Pi stress in Vibrio cholerae, Pseudomonas spp., and pathogenic E. coli.

PhoB activates Escherichia coli O157:H7 virulence factors in response to inorganic phosphate limitation.

Enterohemorrhagic Escherichia coli (EHEC), an emerging food- and water-borne hazard, is highly pathogenic to humans. In the environment, EHEC must survive phosphate (Pi) limitation. The response to such Pi starvation is an induction of the Pho regulon including the Pst system that senses Pi variation. The interplay between the virulence of EHEC, Pho-Pst system and environmental Pi remains unknown. To understand the effects of Pi deprivation on the molecular mechanisms involved in EHEC survival and virulence under Pho regulon control, we undertook transcriptome profiling of the EDL933 wild-type strain grown under high Pi and low Pi conditions and its isogenic ΔphoB mutant grown in low Pi conditions. The differentially expressed genes included 1067 Pi-dependent genes and 603 PhoB-dependent genes. Of these 131 genes were both Pi and PhoB-dependent. Differentially expressed genes that were selected included those involved in Pi homeostasis, cellular metabolism, acid stress, oxidative stress and RpoS-dependent stress responses. Differentially expressed virulence systems included the locus of enterocyte effacement (LEE) encoding the type-3 secretion system (T3SS) and its effectors, as well as BP-933W prophage encoded Shiga toxin 2 genes. Moreover, PhoB directly regulated LEE and stx2 gene expression through binding to specific Pho boxes. However, in Pi-rich medium, constitutive activation of the Pho regulon decreased LEE gene expression and reduced adherence to HeLa cells. Together, these findings reveal that EHEC has evolved a sophisticated response to Pi limitation involving multiple biochemical strategies that contribute to its ability to respond to variations in environmental Pi and to coordinating the virulence response.

The ecological habitat and transmission of Escherichia coli O157:H7.

Since its first description in 1982, the zoonotic life-threatening Shiga toxin-producing Escherichia coli O157:H7 has emerged as an important food- and water-borne pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. In the last decade, increases in E. coli O157:H7 outbreaks were associated with environmental contamination in water and through fresh produce such as green leaves or vegetables. Both intrinsic (genetic adaptation) and extrinsic factors may contribute and help E. coli O157:H7 to survive in adverse environments. This makes it even more difficult to detect and monitor food and water safety for public health surveillance. E. coli O157:H7 has evolved in behaviors and strategies to persist in the environment.

Survival of enterohemorrhagic Escherichia coli in the presence of Acanthamoeba castellanii and its dependence on Pho regulon.

Enterohemorrhagic Escherichia coli (EHEC) are involved in outbreaks of food-borne illness and transmitted to humans through bovine products or water contaminated by cattle feces. Microbial interaction is one of the strategies used by pathogenic bacteria to survive in the environment. Among protozoa, the free-living amoebae are known to host and protect several water-borne pathogens. In this study, the interaction between EHEC and the predacious protozoa Acanthamoeba castellanii was investigated. Using monoculture and cocultures, growth of both organisms was estimated for 3 weeks by total and viable cell counts. The numbers of EHEC were significantly higher when cultured with amoebae than without, and less EHEC shifted into a viable but nonculturable state in the presence of amoebae. Using several mutants, we observed that the Pho regulon is required for EHEC growth when cocultured with amoebae. In contrast, the Shiga toxins (Stx) were not involved in this association phenotype. Cocultures monitored by electron microscopy revealed a loss of the regular rod shape of EHEC and the secretion of multilamellar vesicles by the amoebae, which did not contain bacteria. As the interaction between A. castellanii and EHEC appears beneficial for bacterial growth, this supports a potential role for protozoa in promoting the persistence of EHEC in the environment.

The Pho regulon and the pathogenesis of Escherichia coli.

During the course of infection, bacteria must coordinately regulate gene expression in response to environmental stimuli. The phosphate (Pho) regulon is controlled by the two component-regulatory system PhoBR. PhoBR is activated during starvation and regulates genes involved in phosphate homeostasis. Several studies have highlighted the importance of the Pho regulon in bacterial pathogenesis, showing how induction of PhoBR, in addition to regulating genes participating in phosphate metabolism, leads to modulation of many cellular processes. The pleiotropic effects of Pho regulon activation include attenuated virulence and alteration of many virulence traits, including adhesion to host cells and resistance to cationic antimicrobial peptides, acidity and oxidative stresses. This review provides an overview of the relationship between the Pho regulon and virulence in Escherichia coli and illustrates that, in addition to regulating phosphate homeostasis, the Pho regulon plays a key role in regulating stress responses and virulence.