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Multimodal Imaging Reveals Spatial Host-Pathogen Microenvironments in Escherichia coli Meningoencephalitis

Dominika LuptákováiD, Tereza JuříkováiD, Oldřích BenadaiD, Gabriela Lokočová, Helena MarešováiD, Hynek Mácha, Jiří HoušťiD, Kateřina Dvořáková BendováiD, Miroslav PopperiD, Miloš PetříkiD, Andrea PalyzováiD, Lukáš KučeraiD, E. N. BiryukovaiD, Jiří NovákiD, Vladimı́r Havlı́čekiD

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Abstract

Experimental bacterial meningitis is often analyzed as microbial burden and inflammation, but the tissue-level organization of host-pathogen interplay remains poorly resolved. Here, we use multimodal imaging to map Escherichia coli meningoencephalitis as a spatially organized process across infected rat brain tissue and cerebrospinal fluid (CSF). A low-dose intracerebral inoculum expands within 24 h into an anatomically structured infection involving ventricular, meningeal, and perivascular interfaces. Scanning electron microscopy reveals biofilm-like multicellular E. coli aggregates embedded in extracellular material, consistent with neutrophil extracellular traps. MALDI mass spectrometry imaging detects aerobactin and salmochelin-derived metabolites, whereas intact enterobactin is not detected despite favorable analytical sensitivity. These bacterial iron-acquisition signals partially overlap with calprotectin proteoforms, defining ventricular and periventricular metal-conflict microenvironments. Spatial peptidomics identifies infection-enriched antimicrobial territories dominated by rat neutrophil peptides RatNP-2, RatNP-3, and RatNP-4. Endogenous proenkephalin-derived neuropeptides are reduced in basal ganglia regions. Finally, cerebrospinal fluid captures both bacterial siderophores and host antimicrobial peptides with lipocalin-2 protein, linking tissue-resolved host-pathogen chemistry to a proximal diagnostic fluid. Together, these data show that experimental E. coli meningoencephalitis is a spatially organized host-pathogen process. Bacterial communities, nutritional immunity, antimicrobial peptides, and neuropeptide networks occupy distinct but connected CNS niches, components of which are recoverable in CSF.

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 7 Nov 2026 · from the full text of version 2 (31 Jul 2026) · CC BY 4.0

Rat E. coli meningoencephalitis maps into ventricular, meningeal and perivascular niches: biofilm-like aggregates, aerobactin and salmochelin signals, calprotectin, neutrophil peptides, reduced proenkephalin peptides, with some markers recoverable in CSF.

The paper says bacterial burden and inflammatory mediators, averaged across tissue, cannot show how host and pathogen molecules are arranged in infected brain. It combines scanning electron microscopy (SEM), MALDI mass spectrometry imaging (MSI) on serial sections of the same brains, and cerebrospinal fluid (CSF) analysis to map this.

Key results

  1. SuggestedA low-dose intracerebral inoculum gave a 24 h infection with meningeal, perivascular and ventricular inflammation. SEM showed biofilm-like E. coli aggregates among fibers the authors read as neutrophil extracellular trap (NET)-like.

    Burden reached up to 1.4×10^5 genome equivalents/mg by qPCR and 6.1×10^3 CFU/mg by culture. Leptomeningeal inflammation P < 0.0001, perivascular P = 0.0003 vs saline controls. SEM fibers about 200 nm and about 20 nm thick; SEM used n = 2 infected brains. · Low-dose inoculum E. coli infection establishes…; Scanning electron microscopy reveals…; Discussion

  2. ShownMALDI MSI detected aerobactin as the main siderophore signal, plus salmochelin SX and 2,3-dihydroxybenzoylserine (DHBS) in infected tissue. Enterobactin was not detected. Siderophores partly overlapped with calprotectin proteoforms.

    DHBS vs S100A9-ac/m at +2.28 mm: ρ = 0.78, P = 0.0048, FDR q = 0.0149; DHBS vs S100A8 at -1.08 mm: ρ = 0.52, FDR q = 0.0171. Lcn-2 was seen only in infected tissue. Infected n = 5, control n = 4. · Bacterial siderophores and host calprotectin…

  3. ShownUnsupervised peptidomics found infection-enriched territories dominated by rat neutrophil peptides RatNP-2, -3 and -4, concentrated at ventricular, meningeal and limbic regions. These peptides tracked calprotectin more closely than siderophores.

    RatNPs were 181- to 226-fold higher in infected brains at +2.28 mm and 860- to 2303-fold at -1.08 mm. RatNP-3 reached up to 30.6 µg/g. RatNP-2 vs S100A9-ac: ρ = 0.93, FDR q = 0.0033 (+2.28 mm). Six UMAP/k-means clusters from 111 features. · Unsupervised spatial peptidomics identifies…; Rat neutrophil peptides define ventricular and…

  4. ShownProenkephalin-derived peptides were coordinately lower in infected basal ganglia regions than in controls.

    Ten proenkephalin peptides annotated. Total PEnk signal 49% lower (P = 0.0293, Welch's t-test). PEnk (143-185) P = 0.0148 and PEnk (219-229) P = 0.0051, a 47% decrease. Infected n = 5, control n = 4. · E. coli meningoencephalitis reduces the basal…

  5. SuggestedCSF from infected rats contained RatNP-2, -3, -4, Lcn-2 and several E. coli siderophores, mirroring the tissue pattern. Enterobactin was again absent.

    These analytes were undetectable in control CSF. Aerobactin was the most abundant siderophore, with salmochelins S1, S2, S5 and SX. MALDI MS, ELISA and LC-MS on n = 5 infected vs n = 4 control (Lcn-2 ELISA controls n = 8); no test statistics given. · CSF captures host antimicrobial peptides with…; LC-MS analysis of siderophores in CSF and brain…; MALDI MS analysis of antimicrobial peptides in…; Analysis of rat lipocalin-2 in infected and…

Methods, data and reproducibility

E. coli CCM 5172 or saline was injected intracerebrally into rats. Brains were analyzed at 24 h by histology, qPCR, culture, SEM and MALDI MSI of siderophores, peptides and proteins at two coronal levels. CSF was analyzed by MALDI MS, LC-MS and ELISA.

  • Female Lewis rats, three months old, E. coli-infected vs saline control; MSI, qPCR, culture, histology and CSF analyses · Rat · n = Infected n = 5, control n = 4 (inoculated: 7 low-dose, 7 higher-dose, 6 saline)
  • SEM of float-fixed brain sections · Rat · n = Infected n = 2, control n = 2
  • Raw mass spectrometry data (MALDI MSI, LC-MS) · Rat · 10.57680/asep.0651117
Methods & tools
MALDI FTICR MSI (solariX 12T-2ω), MALDI-TOF/TOF (Ultraflex II), SCiLS Lab v.2026b Pro, mMass, UMAP with bisecting k-means, CycloBranch 2.1.35, LC-MS, SEM (FEI Nova NanoSEM 450), RayBio rat Lipocalin-2 ELISA, GraphPad Prism 10.0.1
Software & environment
SCiLS Lab v.2026b Pro, CycloBranch 2.1.35, DataAnalysis 6.0, CFX Manager v3.1, GraphPad Prism 10.0.1, ImageJ.
Reproducibility gaps
No analysis scripts are stated; the code link is the CycloBranch viewer site. Remaining data are said to be in the main text or supplement. Some in vitro data are marked 'data not shown'.
Limitations (6)
  • Direct intracerebral inoculation bypassed bloodstream invasion and blood-brain barrier crossing. · stated by the authors
  • A single 24 h endpoint captured spatial organization but not the temporal sequence of the response. · stated by the authors
  • Sample numbers were limited in a controlled model, so validation in etiologically confirmed clinical meningitis is needed before any diagnostic claim. · stated by the authors
  • Biofilm matrix composition and causal relationships between the mapped molecules were not defined; the proenkephalin mechanism is hypothesized and would need transcript or receptor analysis. · stated by the authors
  • The NET-like assignment of SEM fibers rests on fiber diameter and morphology from only 2 infected brains, with no matrix-specific staining. · noted in this summary
  • Only one E. coli strain (CCM 5172) and one rat strain and sex (female Lewis) were studied, so generality is untested. · noted in this summary

Read this if you work on spatial mass spectrometry imaging of infected tissue and want a worked example linking bacterial siderophores, host nutritional immunity and neutrophil peptides to CSF markers.

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