The morphology of macrophages changes following infection with <i>R. equi</i>.

<p>J774A.1 monolayers were infected with ATTO 488 labelled virulent <i>R. equi</i> 103+ (green). Monolayers were fixed 24 h post-infection; the actin cytoskeleton and cell nuclei were stained with Texas Red Phalloidin (red) and Hoechst 33258 (blue), respectively. Panel A: non-infected monolayers, panel B: monolayers infected with <i>R. equi</i> 103+. Arrows indicate the change in cell shape following infection with <i>R. equi</i>. The length of the white bar is 40 µm.</p

Bacterial strains, plasmids and oligonucleotides used in this study.

<p>Bacterial strains, plasmids and oligonucleotides used in this study.</p

Intracellular growth of virulent and attenuated <i>R. equi</i>.

<p>J774A.1 monolayers were infected with virulent (♦, <i>R. equi</i> 103+; ∇, <i>R. equi</i> Δ35000) and attenuated (□, <i>R. equi</i> 103−; •, <i>R. equi</i> Δ<i>vapA</i>) <i>R. equi</i> strains. Following a 1 hour incubation to allow phagocytosis, monolayers were washed and treated with vancomycin to kill remaining extra cellular bacteria. Intracellular bacteria were enumerated via real time qPCR of the <i>R. equi</i> 16S rRNA gene. Results are expressed as fold change in bacterial numbers relative to t = 0 (h) values. Error bars denote the standard deviation of the mean.</p

Time-dependent Cell Response Profile (TCRP) of infected macrophage monolayers.

<p>Monolayers were infected with a virulent (<i>R. equi</i> 103+; top panel) or with an avirulent (<i>R. equi</i> 103−; bottom panel) strain of <i>R. equi</i> at increasing multiplicities (MOI) of infection. The TCRP was normalized against the TCRP of a non-infected macrophage monolayer.</p

Key TCRP parameters of macrophage monolayers infected with virulent or attenuated <i>R. equi</i> strains.

<p>Shown are the maximum cell indices (Ci) values (panel A) and the the C_i inflection times (panel B) following infection of J774A.1 macrophage monolayers with virulent (♦, <i>R. equi</i> 103+; ∇, <i>R. equi</i> Δ35000) and attenuated (□, <i>R. equi</i> 103−; •, <i>R. equi</i> Δ<i>vapA</i>) <i>R. equi</i> strains at increasing multiplicity of infection. Values are the means ± SD.</p

Infection of macrophages with <i>R. equi</i> affects host cell size and morphology.

<p>Macrophage monolayers were infected with ATTO 488 labeled virulent <i>R. equi</i> 103+ or attenuated <i>R. equi</i> 103− strains at increasing multiplicities (MOI) of infection. Monolayers were fixed and labelled 24 h post-infection. The cell size (panel A) and cell roundness (panel B) of infected cells were compared to control cells (not infected, indicated as MOI = 0). Green bars: no intracellular bacteria; blue bars: 1 to 5 intracellular bacteria; red bars: more than 5 intracellular bacteria. +: <i>R. equi</i> 103+; −: <i>R. equi</i> 103−. Error bars denote the standard deviation of the mean.</p

Macrophages in infected monolayers are not homogeneous with respect to the number of intracellular <i>R. equi</i> they harbour.

<p>Macrophage monolayers were infected with ATTO 488 labeled virulent (<i>R. equi</i> 103+; top panel) or attenuated (<i>R. equi</i> 103−; bottom panel) strains of <i>R. equi</i> at increasing multiplicities (MOI) of infection. Monolayers were fixed and labelled 24 h post-infection. High content image analysis using Columbus software was employed to enumerate infected macrophages and to determine the number of intracellular <i>R. equi</i>. Shown are the number of macrophages that contained no intracellular bacteria (grey), between 1 and 5 bacteria (white) or more than 5 bacteria (black).</p

Addition of a C-terminal Strep-tag does not affect cell surface localisation of VapA.

R. equi ΔvapA/pVapA-ST was grown under vapA inducing conditions, followed by extraction with 2% (v/v) Triton X-114. VapA monoclonal antibodies and Strep-tag HRP conjugate antibodies were used to detect VapA. Lane 1: western blot developed using VapA monoclonal antibodies. Lane 2: western blot developed using Strep-tag HRP conjugate antibodies. Bars on the left indicate the molecular mass in kDa.</p

Flow cytometry determination of VapA cell surface localization in <i>Rhodococcus equi</i> using a C-terminal Strep-tagged VapA.

VapA-ST is expressed as a cell surface protein. Flow cytometry of R. equi ΔvapA carrying either pVapA-ST (A, C and D) or pVapA (B). Approximately 7x105 cells were incubated with THE2122; NWSHPQFEK Tag mouse FITC-monoclonal antibody (A, B and D). Cells carrying pVapA were used to assess the level of antibody’s non-specific binding (B). The intrinsic fluorescence of R. equi ΔvapA/pVapA-ST was determined by including a control incubated without the antibody (C). The bacterial sample was digested with Trypsin to remove proteins from the cell surface before incubation with antibody (D). Data are a representative of three independent experiments.</p

The N-terminus of VapA plays a role in surface localization.

Graphical representation of the fusion proteins containing the N-terminus of VapA (T32-Q84) and the C-terminal domain of VapD that were employed to assess the role of the N-terminus of VapA on surface localization (A). Fusion proteins VapA(SS-N)::VapD-ST (B) and VapD(SS)::VapA(N)::VapD-ST (C). Cell surface proteins were detected with THE2122; NWSHPQFEK Tag mouse FITC-monoclonal antibody (Red) but not detected when cells were digested with trypsin before incubation with the antibody (Black).</p