Community behavior and spatial regulation within a bacterial microcolony in deep tissue sites serves to protect against host attack

Abstract

Bacterial pathogens express virulence-specific transcriptional programs that allow tissue colonization. Although phenotypic variation has been noted in the context of antibiotic exposure, no direct evidence exists for heterogeneity in virulence-specific transcriptional programs within tissues. In a mouse model of Yersinia pseudotuberculosis infection, we show that at least three subpopulations of bacteria develop within a single tissue site in response to distinct host signals. Bacteria growing on the exterior of spleen microcolonies responded to soluble signals and induced the nitric oxide (NO)-detoxifying gene, hmp. Hmp effectively eliminated NO diffusion and protected the interior bacterial population from exposure to NO-derived inducing signals. A third subpopulation, constituting the most peripherally localized bacteria, directly contacted neutrophils and transcriptionally upregulated a virulence factor. These studies demonstrate that growth within tissues results in transcriptional specialization within a single focus of microbial replication, facilitating directed pathogen counterattack against the host response.

Figure 1. Spatial control of Y. pseudotuberculosis gene expression during growth in tissues

C57BL/6 mice were inoculated intravenously with 10³ WT Y. pseudotuberculosis, and spleens were harvested at day 3 post-inoculation (PI). A) Inoculated with an equal mixture of Yptb GFP⁺ (P_tet::gfp expressed from pACYC184, lacking tetO) and Yptb mCherry⁺ (yopE::mCherry, integrated downstream of yopE⁺). Sections stained with Hoechst and visualized by fluorescence microscopy. 7 mice were analyzed, with 3–11 centers/mouse. B) Three distinct microcolonies (arrows), low power magnification. C) Low and high power magnification of H&E stained sections from a mouse infected with WT, centered on area of inflammation (dashed box, I). R: red pulp region, I: area of inflammation. D) Bacterial transcripts quantified by qRT-PCR. Values expressed relative to 16S. Each bar represents an individual mouse (numbered, black bars) or the inoculum (white bars). E) Nos2 transcripts quantitified by qRT-PCR, relative to Gapdh, and relative to uninfected controls. Each bar represents an individual mouse (numbered), and corresponds to the mice in panel D. F) Mice inoculated with the WT gfp⁺ P_hmp::mCherry strain. Frozen splenic sections stained with Hoechst to detect host nucleic acids. Ratiometric image of the mCherry (P_hmp) signal/GFP signal (range: .2–2) displayed using rainbow scale (Experimental Procedures). See also Figure S1.

Figure 2. Hmp activity is required for spatial regulation of P_hmp

C57BL/6 mice intravenously inoculated with WT gfp⁺ P_hmp::mCherry or Δhmp gfp⁺ P_hmp::mCherry strains, and spleens harvested at day 3 PI. Frozen sections prepared, and bacterial reporter expression visualized by fluorescence microscopy. A) Size-matched representative images of four WT and four Δhmp microcolonies. B) Colorimetric depiction of signal intensity (scale bars) in each channel (top: mCherry, bottom: GFP) from the bottom images in (A). C) hmp(mCherry)/GFP ratios generated by dividing signal intensity of each channel at the centroid and periphery of each replication center. Each dot represents an individual microcolony, 2–12 centers analyzed/mouse, 5 mice. The dotted line: signal intensity equivalent for each channel. D) Δhmp rescued by integrating hmp::mCherry construct into chromosome. Representative microcolony shown alongside hmp(mCherry)/GFP ratios, generated as described in C. 1–10 centers analyzed/mouse, 5 mice. Statistics: Mann-Whitney (comparison between centroids), Wilcoxon matched pairs (comparison between centroid and periphery), ***p<0.001, n.s.: not significant. Scale bars: 20μm. See also Figure S2.

Figure 3. Distinct subpopulations of peripheral bacteria within microcolonies

A) Increasing magnifications of area of inflammation (I, dashed box) from splenic section, stained with H&E. W: white pulp region, R: red pulp region, I: area of inflammation. B) C57BL/6 mice intravenously inoculated with the WT gfp⁺ yopE::mCherry or Δhmp gfp⁺ yopE::mCherry strains, and spleens harvested at day 3 PI. Frozen sections prepared and visualized by fluorescence microscopy. C) yopE(mCherry)/GFP ratios generated as described in Figure 2C. 1–11 centers analyzed/mouse, 5 mice/infection. D) C57BL/6 mice intravenously inoculated with WT P_hmp::gfp yopE::mCherry or Δhmp P_hmp::gfp yopE::mCherry strains, and spleens harvested at day 3 PI. Frozen sections prepared and visualized by fluorescence microscopy. Representative images shown with colorimetric depiction of signal intensity for each channel. E) hmp(GFP)/yopE(mCherry) ratios generated as described in Figure 2C. 2–11 centers analyzed/mouse, 6 mice/infection. F) Nitrogen stress genes induced with NO₂ (NO₂, +), and/or type-III secretion system genes induced by low Ca²⁺ conditions (T3SS, +), in cultures of the WT strain. Transcription levels expressed relative to 16S. Each bar represents four replicates, error bars depict the median and range. Statistics: Mann-Whitney (in vitro transcription), Wilcoxon matched pairs (for comparison between centroid and periphery), ***p<0.001, ** p<0.01, *p<0.05, n.s.: not significant.

Figure 4. Bacteria do not directly contact iNOS producing host cells

A) Nos2^−/− mice infected with WT gfp⁺ P_hmp::mCherry strain at day 3 PI. Spleen frozen sections prepared, stained with antibodies for indicated host markers and visualized by fluorescence microscopy. 23 centers were analyzed across 2 mice. B) C57BL/6 mice intravenously inoculated with WT (black bars, 5 mice), or PBS (white bars, 3 mice). Splenocytes were detected by flow cytometry. Values represent % of each cell type within total population of iNOS⁺ cells. C) Splenocytes stained and detected by flow cytometry. Values represent % of iNOS⁺ cells within each cell type. D) Low and high power magnification of an area of inflammation (dashed box, I), from splenic section of a mouse infected with WT. W: white pulp region, R: red pulp region, I: area of inflammation. E) & F) C57BL/6 mice intravenously inoculated with GFP⁺ WT strain, splenic frozen sections prepared at day 3 PI, and stained with antibodies. E) Low power magnification. F) High power magnification. Inset: zoom of colony interface. G) C57BL/6 mice intravenously inoculated with GFP⁺ WT, splenic sections prepared at day 3 PI, and antibody stained. Nine representative images shown. Panels A, E, F, & G all depict distinct microcolonies, 115 microcolonies were analyzed from 14 mice. See also Figure S3.

Figure 5. The absence of hmp causes reduced fitness

A) Balb/c mice intravenously inoculated with the GFP⁺ WT or GFP⁺ Δhmp strain, and spleens harvested at days 3 and 5 PI. Microcolony areas (μm²) were quantified (Experimental Procedures) in 5 mice, with 6–10 total centers/mouse. B) WT and Δhmp microcolonies at day 5 PI. C) C57BL/6 mice intravenously inoculated with an equal mixture of mCherry⁺ (yopE::mCherry) WT and GFP⁺ Δhmp strains, and spleens harvested at day 3 PI. Competitive index: ratio of Δhmp/WT spleen CFUs at day 3, divided by the ratio of Δhmp/WT CFUs in inoculum. Each dot: individual mouse. Dotted line: each strain has equal fitness. WT and mutant microcolony areas (μm²), quantified (Experimental Procedures) in 7 mice, with 6–10 total centers/mouse. D) Representative WT and Δhmp microcolonies from same organ shown at day 3 PI. Statistics: Mann-Whitney, ***p<0.001, **p<0.01. Scale bars: 50μm. See also Figure S4.

Figure 6. Bacteria within microcolonies fail to activate post-exponential genes

A) C57BL/6 mice intravenously inoculated with WT strain and spleens harvested at day 3 PI. Bacterial RNA isolated, reverse transcribed, and transcripts detected by qRT-PCR. Transcription levels expressed relative to 16S. Bars: individual mice, or mean of culture condition (6 replicates/culture condition). B) C57BL/6 mice intravenously inoculated with WT P_katG::gfp yopE::mCherry strain, and spleens harvested at day 3 PI. Frozen sections prepared and visualized by fluorescence microscopy. 2–8 centers analyzed/mouse, 5 mice. Three representative images are shown. Scale bars: 20μm. See also Figure S5.