Kinetic Characterization of the Immune Response to Methicillin-Resistant Staphylococcus aureus Subcutaneous Skin Infection.

Staphylococcus aureus is a leading cause of skin and soft tissue infections (SSTIs). Studies examining the immune response to S. aureus have been conducted, yet our understanding of the kinetic response to S. aureus subcutaneous skin infection remains incomplete. In this study, we used C57BL/6J mice and USA300 S. aureus to examine the host-pathogen interface from 8 h postinfection to 15 days postinfection (dpi), with the following outcomes measured: lesion size, bacterial titers, local cytokine and chemokine levels, phenotype of the responding leukocytes, and histopathology and Gram staining of skin tissue. Lesions were largest at 1 dpi, with peak necrotic tissue areas at 3 dpi, and were largely resolved by 15 dpi. During early infection, bacterial titers were high, neutrophils were the most abundant immune cell type, there was a decrease in most leukocyte populations found in uninfected skin, and many different cytokines were produced. Histopathological analysis demonstrated swift and extensive keratinocyte death and robust and persistent neutrophil infiltration. Gram staining revealed subdermal S. aureus colonization and, later, limited migration into upper skin layers. Interleukin-17A/F (IL-17A/F) was detected only starting at 5 dpi and coincided with an immediate decrease in bacterial numbers in the following days. After 9 days, neutrophils were no longer the most abundant immune cell type present as most other leukocyte subsets returned, and surface wounds resolved coincident with declining bacterial titers. Collectively, these data illustrate a dynamic immune response to S. aureus skin infection and suggest a key role for precisely timed IL-17 production for infection clearance and healthy tissue formation.

The Small RNA Teg41 Is a Pleiotropic Regulator of Virulence in Staphylococcus aureus.

Previously, our group demonstrated a role for the small RNA (sRNA) Teg41 in regulating production of the alpha phenol-soluble modulin toxins (αPSMs) in Staphylococcus aureus. Overexpressing Teg41 increased αPSM production while deleting the 3' end of Teg41 (Teg41Δ3' strain) resulted in a decrease in αPSM production, reduced hemolytic activity of S. aureus culture supernatants, and attenuated virulence in a murine abscess model of infection. In this study, we further explore the attenuation of virulence in the Teg41Δ3' strain. Using both localized and systemic models of infection, we demonstrate that the Teg41Δ3' strain is more severely attenuated than an ΔαPSM mutant, strongly suggesting that Teg41 influences more than the αPSMs. Proteomic and transcriptomic analysis of the wild-type and Teg41Δ3' strains reveals widespread alterations in transcript abundance and protein production in the absence of Teg41, confirming that Teg41 has pleiotropic effects in the cell. We go on to investigate the molecular mechanism underlying Teg41-mediated gene regulation. Surprisingly, results demonstrate that certain Teg41 target genes, including the αPSMs and ßPSMs, are transcriptionally altered in the Teg41Δ3' strain, while other targets, specifically spa (encoding surface protein A), are regulated at the level of transcript stability. Collectively, these data demonstrate that Teg41 is a pleiotropic RNA regulator in S. aureus that influences expression of a variety of genes using multiple different mechanisms.

YjbH contributes to Staphylococcus aureus skin pathology and immune response through Agr-mediated α-toxin regulation.

Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs) with Methicillin-Resistant S. aureus (MRSA) strains being a major contributor in both community and hospital settings. S. aureus relies on metabolic diversity and a large repertoire of virulence factors to cause disease. This includes α-hemolysin (Hla), an integral player in tissue damage found in various models, including SSTIs. Previously, we identified a role for the Spx adapter protein, YjbH, in the regulation of several virulence factors and as an inhibitor of pathogenesis in a sepsis model. In this study, we found that YjbH is critical for tissue damage during SSTI, and its absence leads to decreased proinflammatory chemokines and cytokines in the skin. We identified no contribution of YjbI, encoded on the same transcript as YjbH. Using a combination of reporters and quantitative hemolysis assays, we demonstrated that YjbH impacts Hla expression and activity both in vitro and in vivo. Additionally, expression of Hla from a non-native promoter reversed the tissue damage phenotype of the ΔyjbIH mutant. Lastly, we identified reduced Agr activity as the likely cause for reduced Hla production in the ΔyjbH mutant. This work continues to define the importance of YjbH in the pathogenesis of S. aureus infection as well as identify a new pathway important for Hla production.

The Staphylococcus aureus small non-coding RNA IsrR regulates TCA cycle activity and virulence.

Staphylococcus aureus has evolved mechanisms to cope with low iron (Fe) availability in host tissues. S. aureus uses the ferric uptake transcriptional regulator (Fur) to sense titers of cytosolic Fe. Upon Fe depletion, apo-Fur relieves transcriptional repression of genes utilized for Fe uptake. We demonstrate that an S. aureus Δfur mutant has decreased expression of acnA, which codes for the Fe-dependent enzyme aconitase. Decreased acnA expression prevented the Δfur mutant from growing with amino acids as sole carbon and energy sources. Suppressor analysis determined that a mutation in isrR, which produces a regulatory RNA, permitted growth by decreasing isrR transcription. The decreased AcnA activity of the Δfur mutant was partially relieved by an ΔisrR mutation. Directed mutation of bases predicted to facilitate the interaction between the acnA transcript and IsrR, decreased the ability of IsrR to control acnA expression in vivo and IsrR bound to the acnA transcript in vitro. IsrR also bound to the transcripts coding the alternate TCA cycle proteins sdhC, mqo, citZ, and citM. Whole cell metal analyses suggest that IsrR promotes Fe uptake and increases intracellular Fe not ligated by macromolecules. Lastly, we determined that Fur and IsrR promote infection using murine skin and acute pneumonia models.