Intravital two-photon microscopy of host-pathogen interactions in a mouse model ofStaphylococcus aureusskin abscess formation

Abstract

SummaryStaphylococcus (S.) aureus is a frequent cause of severe skin infections. The ability to control the infection is largely dependent on the rapid recruitment of neutrophils (PMN). To gain more insight into the dynamics of PMN migration and hostpathogen interactions in vivo, we used intravital two-photon (2-P) microscopy to visualize S. aureus skin infections in the mouse. Reporter S. aureus strains expressing fluorescent proteins were developed, which allowed for detection of the bacteria in vivo. By employing LysM-EGFP mice to visualize PMN, we observed the rapid appearance of PMN in the extravascular space of the dermis and their directed movement towards the focus of infection, which led to the delineation of an abscess within 1 day. Moreover, tracking of transferred labelled bone-marrow neutrophils showed that PMN localization to the site of infection is dependent on the presence of G-protein-coupled receptors on the PMN, whereas Interleukin-1 receptor was required on host cells other than PMN. Furthermore, the S. aureus complement inhibitor Ecb could block PMN accumulation at the site of infection. Our results establish that 2-P microscopy is a powerful tool to investigate the orchestration of the immune cells, S. aureus location and gene expression in vivo on a single cell level.

Local and global consequences of flow on bacterial quorum sensing

“…A plasmid constitutively expressing mKate under the sarA P1 promotor (pMK014), was constructed by replacing the agr P3 promoter in pMK004 with the constitutive promoter sarA P1. The sarA P1 sequence corresponded to −306 to −62 upstream of sarA , and was amplified from S. aureus RN6390b using primers MKF001/MKR001 10,11,33 , followed by overlap extension PCR cloning. The final plasmid harboring both the agr P3- gfpmut2 and the sarA P1- mKate fusions (pMK021) was constructed by introducing the sarA P1- mKate fragment onto pJL-agr-GFP using Gibson cloning (New England Biolabs, Ipswich, MA) 34 .…”

“…The S. aureus strain harbored an agr P3- gfpmut2 transcriptional fusion 11 , which is activated in response to the endogenously produced QS autoinducer called AIP-I 2 (Supplementary Fig. 1a), and a constitutively expressed sarA P1- mKate transcriptional fusion 12 , which enabled us to normalize measurements of QS responses by correcting for the effects of growth (Supplementary Fig.…”

Aging‐related anatomical and biochemical changes in lymphatic collectors impair lymph transport, fluid homeostasis, and pathogen clearance

“…For imaging bacterial transport through the lymphatic collectors, young and aged mice were injected with 1 × 10 8 CFU of S. aureus expressing GFP under the control of the endogenous Agr promoter (a gift from Jan Liese) (Liese et al ., 2013). To visualize the lymphatic, 66 kD dextran (5 μg, Sigma) conjugated in-house to trimethylrhodamine (TRITC) was synthesized and purified as described previously (Proulx et al ., 2013) by conjugating a 20 kDa methoxypoly (ethylene glycol) amine (Sigma) to IRDye® 680LT NHS ester (LICOR Biosciences, Lincoln, NE, USA).…”

Fluorescence Reporter in Staphylococcus aureus as a Useful Tool for Studying L-forms and Virulence

“…The bac- terial quantification in lesions was calculated by analyzing the pixels in Gram-positive areas and bacterial photon intensity. Liese et al used fluorescence labeled S. aureus to visualize bacterial skin infections in mice (29). In our study, we extended the fluorescence reporter technique to fluorescence intensity measurements and bacterial quantification at the time of virulence determination.…”