Differentiating Staphylococcus aureus from Escherichia coli mastitis: S. aureus triggers unbalanced immune-dampening and host cell invasion immediately after udder infection

Abstract

The etiology determines quality and extent of the immune response after udder infection (mastitis). Infections with Gram negative bacteria (e.g. Escherichia coli) will quickly elicit strong inflammation of the udder, fully activate its immune defence via pathogen receptor driven activation of IκB/NF-κB signaling. This often eradicates the pathogen. In contrast, Gram-positive bacteria (e.g. Staphylococcus aureus) will slowly elicit a much weaker inflammation and immune response, frequently resulting in chronic infections. However, it was unclear which immune regulatory pathways are specifically triggered by S. aureus causing this partial immune subversion. We therefore compared in first lactating cows the earliest (1-3 h) udder responses against infection with mastitis causing pathogens of either species. Global transcriptome profiling, bioinformatics analysis and experimental validation of key aspects revealed as S. aureus infection specific features the (i) failure to activating IκB/NF-κB signaling; (ii) activation of the wnt/β-catenin cascade resulting in active suppression of NF-κB signaling and (iii) rearrangement of the actin-cytoskeleton through modulating Rho GTPase regulated pathways. This facilitates invasion of pathogens into host cells. Hence, S. aureus mastitis is characterized by eliciting unbalanced immune suppression rather than inflammation and invasion of S. aureus into the epithelial cells of the host causing sustained infection.

Figure 1

Number and time course of differentially activated genes during E. coli and S. aureus mastitis and their contribution to regulatory pathways. Venn diagrams of (A) total number of loci regulated either by E. coli or S. aureus infection and (B) number of loci regulated at 1, 2 or 3 h pi during either E. coli or S. aureus infection. (C) Ingenuity based identification of key signaling pathways as regulated by either E.coli or S. aureus infection or by both pathogens. The ordinate indicates the logarithm of the statistical significance (p-value). The z-score represents the likelihood that the pathway is regulated (red, up-; blue, down-regulated), with more intense colors reflecting higher significance. Ratio indicates the proportion of DEGs found in the current data set relative to the total number of genes contributing to that pathway.

Figure 2

Ingenuity based identification of Upstream Regulators. (A) List and statistical significance of the 3 top-scoring upstream regulators as identified exclusively after E. coli or S. aureus infection or being commonly active during infection with both pathogens. (B) Graphical display of the regulatory networks governing the response towards an S. aureus infection (left) or that with both pathogens (right). Red and green underlay indicate up- or downregulation, respectively, while yellow highlights the up-stream regulators. Figure S2 shows the graphical display of the highly complex regulatory network during an E. coli infection.

Figure 3

S. aureus invasion alters structure of the actin-cytoskeleton. Actin-fibers were stained with Alexa-Fluor tagged phalloidin from (a) control pbMEC cultures or (b) after 1 h co-culture with live E. coli ₁₃₀₃ or (c) GFP-tagged S. aureus ₁₀₂₇ pathogens. Images from control and E. coli co-cultured cells were dominated by short, thick ventral actin-stress fibers, mostly terminated by knob-forming focal adhesion foci. These were not evident in cells harboring the green fluorescent S. aureus pathogens. Instead, the actin fibers appeared more filamentous without apparent adhesion foci. (Scale bar, 10 µm).

Figure 4

Summary of the regulatory events triggered by the S. aureus infection. Red and green colors symbolize activation or repression, respectively. BORG: binder of Rho GTPases (here: the DEG CDC42EP3); C1QTNF3, C1q and tumor necrosis factor related protein 3; CTHRC1, collagen triple helix repeat containing 1; DAAM-1, disheveled associated activator of morphogenesis 1; DVL, disheveled; FZD, frizzled receptor; LGR4/5, leucine-rich repeat-containing G-protein coupled receptor 4/5; LIMK, Lim kinase; MLC: myosin light chain; MLCK, myosin light chain kinase; RHOA, ras homolog family member A; RND3, Rho family GTPase 3; ROCK, Rho kinase; RSPO3, r-spondin 3; SYX, pleckstrin homology and RhoGEF domain containing G5.

Figure 5

Graphical summary of the key differences between the early events after E. coli vs S. aureus udder infection. E. coli (yellow) activates in the epithelial cells TLR signaling triggering synthesis and secretion of proinflammatory factors (cyto- and chemokines, triangles), recruiting and activating cellular factors of innate immunity through activating cytokine receptor (CR) signaling. Also immune dampening factors are activated (blue diamond) but, on balance inflammation (flame) outweighs immune dampening (blue water drop). Knobbed red lines symbolize the ventral fibers terminated by focal adhesion plaques. No alteration of the actin cytoskeleton occurs. S. aureus (green Staphylococci) presence is perceived by the epithelial cell through unknown receptors rather than through TLRs or other classical PRRs. This triggers synthesis and secretion of immune dampening factors (blue crescents) surmounting in their effect any proinflammatory stimulation. Moreover, S. aureus is internalized into the epithelial cells, facilitated through rearrangement and interaction with the actin cytoskeleton. This enables pathogen persistence eventually resulting in chronic infection.