Biology subjects

Flannagan, R. S.

Publications and source records attributed to Flannagan, R. S..

3 recordsLinked to original sources

bioRxiv2022

In vivo growth of Staphylococcus lugdunensis is facilitated by the concerted function of heme and non-heme iron acquisition mechanisms

Acquisition of iron underpins the ability of pathogens to cause disease and Staphylococcus lugdunensis has increasingly been recognized as a pathogen that can cause serious infection. In this study, we sought to address the knowledge gap that exists regarding the iron acquisition mechanisms employed by S. lugdunensis, especially during infection of the mammalian host. Here we show that S. lugdunensis utilizes diverse genome encoded iron acquisition mechanisms to satisfy its need for this nutrient. Indeed, S. lugdunensis can usurp hydroxamate siderophores, and staphyloferrin A and B from S. aureus, using the fhuC ATPase-encoding gene. Acquisition of catechol siderophores and catecholamine stress hormones necessitates the presence of the sst-1 transporter-encoding locus, but not the sst-2 locus. Iron-dependent growth in acidic culture conditions necessitates the feoAB locus. Heme iron is acquired via expression of the iron-regulated surface determinant (isd) locus. During systemic infection of mice we demonstrate that while S. lugdunensis does not cause overt illness, it does colonize and proliferate to high numbers in the kidneys. By combining mutations in the various iron acquisition loci, we further demonstrate that only a strain mutated for all of isd, fhuC, sst-1, and feo, versus combination mutants carrying wild type copies of any one of those loci, was attenuated in its ability to proliferate to high numbers in kidneys. Taken together our data reveal that S. lugdunensis requires a repertoire of both heme and non-heme iron acquisition mechanisms to proliferate during systemic infection of mammals. ImportanceAcquisition of iron underpins the ability of pathogens to cause disease and Staphylococcus lugdunensis has increasingly been recognized as a pathogen that can cause serious infection. In this study, we sought to address the knowledge gap that exists regarding the iron acquisition mechanisms employed by S. lugdunensis, especially during infection of the mammalian host. Owing to an inability to synthesize siderophores, growth of S. lugdunensis is dramatically impaired in the presence of transferrin or serum, yet S. lugdunensis nonetheless uses several other genome-encoded iron acquisition mechanisms, in concert, to proliferate within the mammalian host. Therefore, the development of interventions that target bacterial iron acquisition systems should consider the overlapping function of distinct metal acquisition strategies deployed by bacterial pathogens.

microbiology↗

bioRxiv2021

Superantigens promote Staphylococcus aureus bloodstream infection by eliciting pathogenic interferon-gamma (IFNγ) production that subverts macrophage function

Staphylococcus aureus is a foremost bacterial pathogen responsible for a vast array of human diseases. Staphylococcal superantigens (SAgs) constitute a family of potent exotoxins secreted by S. aureus, and SAg genes are found ubiquitously in human isolates. SAgs bind directly to MHC class II molecules and T cell receptors, driving extensive T cell activation and cytokine release. Although these toxins have been implicated in serious disease including toxic shock syndrome, we aimed to further elucidate the mechanisms by which SAgs contribute to staphylococcal pathogenesis during septic bloodstream infections. As most conventional mouse strains respond poorly to staphylococcal SAgs, we utilized transgenic mice encoding humanized MHC class II molecules (HLA-DR4) as these animals are much more susceptible to SAg activity. Herein, we demonstrate that SAgs contribute to the severity of S. aureus bacteremia by increasing bacterial burden, most notably in the liver. We established that S. aureus bloodstream infection severity is mediated by CD4+ T cells and interferon-gamma (IFN{gamma}) is produced to very high levels during infection in a SAg-dependent manner. Bacterial burden and disease severity were reduced by antibody blocking of IFN{gamma}, phenocopying isogenic SAg deletion mutant strains. Additionally, cytokine analysis demonstrated that the immune system was skewed towards a proinflammatory response that was reduced by IFN{gamma} blocking. Infection kinetics and flow cytometry analyses suggested this was a macrophage driven mechanism, which was confirmed through macrophage depletion experiments. Further validation with human leukocytes indicated that excessive IFN{gamma} allowed S. aureus to replicate at a higher rate within macrophages. Together, this suggests that SAgs promote S. aureus survival by manipulating immune responses that would otherwise be effective at clearing S. aureus. This work implicates SAg toxins as critical targets for preventing persistent or severe S. aureus disease.

microbiology↗

bioRxiv2021

Heme-dependent siderophore utilization promotes iron-restricted growth of the Staphylococcus aureus hemB small colony variant

The ability to acquire iron is essential for Staphylococcus aureus to cause infection. Respiration deficient S. aureus small colony variants (SCVs) frequently cause persistent infections, which necessitates they too acquire iron. How SCVs obtain iron remains unknown and so here we addressed this outstanding question by creating a stable hemB mutant in S. aureus USA300 strain LAC. The mutant, auxotrophic for hemin, was assessed for its ability to grow under iron- restriction and with various iron sources. The hemB SCV utilizes exogenously supplied heme but was attenuated for growth under conditions of iron starvation. RNA-seq analyses showed that both WT S. aureus and the hemB mutant sense and respond to iron starvation, however, growth assays show that the hemB mutant is defective for siderophore-mediated iron acquisition. Indeed, the hemB SCV demonstrates limited utilization of endogenous staphyloferrin B or exogenously provided staphyloferrin A, Desferal, and epinephrine, which enabled the SCV to sustain only minimal growth in iron deplete media. Direct measurement of intracellular ATP in hemB and WT S. aureus revealed that both strains can generate comparable levels of ATP during exponential growth suggesting defects in ATP production cannot account for the inability to efficiently utilize siderophores. Defective siderophore utilization by hemB bacteria was also evident in vivo. Indeed, the administration of Desferal failed to promote hemB bacterial growth in vivo, in contrast to WT, in every organ analyzed except for the murine kidney where growth was enhanced. In support of the hypothesis that S. aureus accesses heme in kidney abscesses, in vitro analyses revealed that increased heme availability enables hemB bacteria to utilize siderophores for growth when iron availability is restricted. Taken together, our data support the conclusion that heme is not only used as an iron source itself, but as a nutrient that promotes utilization of siderophore-iron complexes. Author summaryS. aureus small colony variants (SCVs) represent a difficult to treat subpopulation of bacteria that are associated with chronic recurrent infection and worsened clinical outcome. Indeed, SCVs persist within the host despite appropriate administration of antibiotics. This study yields insight into the mechanisms by which S. aureus SCVs acquire iron which, during infection of a host, is a difficult-to-acquire metal nutrient. Under heme limited conditions, hemB S. aureus is severely impaired for siderophore-dependent growth and, in agreement, murine infection indicates that hemin-deficient SCVs meet their nutritional requirement for iron through utilization of heme in vivo. Importantly, we demonstrate that hemB SCVs rely upon heme as a nutrient to promote siderophore utilization. Therefore, perturbation of heme biosynthesis and/or utilization represents a viable to strategy to mitigate the ability of SCV bacteria to acquire siderophore- bound iron during infection.

microbiology↗