Model of the microenvironment of bacterial-induced hemorrhagic lesions. The typical course of non-typhoidal S. enterica infections is shown proceeding through infection, incubation, prodromal, illness, and resolution stages (black arrows). The atypical route of GI bleeding, associated with increased mortality and morbidity, is shown in red arrows, with rates approximated from available literature. An artistic depiction of bacterial injury tropism is shown bottom.

S. enterica serovars rapidly localize toward human serum. A. CIRA experimental design. B. CIRA microgradient model, simulated with a source of 1.13 mM A488 dye after 300 s of injection. C. Visualization of the CIRA microgradient with A488 dye. D. Radial distribution of A488 dye at representative time points (n=6). E. Response of S. enterica Typhimurium IR715 to human serum (max projections over 10 s intervals). F. Quantification of S. enterica Typhimurium IR715 attraction response to human serum (n=4, 37° C) characterized as either the relative number of bacteria within 150 µM of the source (left), or the radial distribution of the bacterial population over time (right, shown in 10 s intervals). G. Area under the curve (AUC) versus time for the bacterial population within 100 µm of the serum treatment source (area indicated in yellow in panel F). Effect size (Cohen’s d) between the treatment start and endpoints is indicated. Insertion of the treatment microcapillary is indicated with black arrow. Attraction rate over time indicated in gray. H-J. CIRA competition experiments between S. Typhimurium IR715 (pink) and clinical isolates (green) responding to human serum for 5 mins (n=4, 37° C). Images are representative max projections over the final minute of treatment. Radial distributions calculated from max projections and averaged across replicates are shown as fold-change relative to the image periphery at 240 µm from the source. Inset plots show fold-change AUC of strains in the same experiment, with p-values from unpaired two-sided t-test, or one-sided t-test (stars) relative to an expected baseline of 1. Trend lines (dashed) indicate the degree of bias in the population distribution, with increasingly negative slope reflecting greater chemoattraction. Data shown are means, error bars indicate SEM. See also Fig. S1, Table S1, Movie S1,

Attraction to human serum is mediated through chemotaxis and the chemoreceptor Tsr. A. Potential mechanisms involved in Salmonella sensing of chemoattractants present in human serum. Approximate concentrations of these effectors in human serum are indicated in parentheses 3. B-F. Microgradient modeling of serum chemoattractant concentrations. G-H. CIRA competition experiments between S. Typhimurium IR715 WT and isogenic mutants cheY, or tsr, and tsr versus cheY, in response to human serum (n=3-4, 37° C). Rates in terms of fold-change are indicated with light pink/light green lines and plotted on the gray secondary y-axis. Data are means and error bars are SEM. See also Table S1, Movie S3.

L-serine is sensed as a chemoattractant molecular cue but provides little growth advantage. A-C. CIRA competition experiments between S. Typhimurium IR715 (pink) and clinical isolates (green) in response to 500 µM L-serine (n=3, 37° C). D. Representative results showing max projections of S. Typhimurium IR715 at 240 – 300 s post CIRA treatment with L-serine concentrations (30° C). E. Quantification of multiple replicate experiments shown in D. F. Attraction and dispersion of S. Typhimurium IR715 following addition and removal of 500 µM L-serine source (30° C). G. S. Typhimurium IR715 WT or tsr mutant responses to L-aspartate or L-serine treatments (30° C). H-I. Serum provides a growth benefit for diverse S. enterica serovars, that is not recapitulated by L-serine treatment alone. Growth is shown as area under the curve (AUC, black) and A600 at mid-log phase for the untreated replicates (gray, n=16). Data are means and error is SEM. See also Fig. S2, Fig. S3, Fig. S4, Movie

Structural mechanism underlying L-serine chemosensing. A. Model of the core chemoreceptor signaling unit showing two full-length Tsr chemoreceptor trimer-of-dimers; coiled-coil region (CC), inner membrane (IM)45. B. Crystal structure of S. enterica Tsr LBD dimer in complex with L-serine (2.2 Å). C-D. Relative order of the SeTsr dimer as indicated by B-factor (Å2). E. Overlay of chains from serine-bound SeTsr (blue), serine-bound EcTsr (orange), and apo EcTsr (white). F. Binding of the L-serine ligand as seen with an overlay of the 5 unique chains of the asymmetric unit (AU) in the SeTsr structure. Purple mesh represents non-crystallographic symmetry 2fo-fc omit map electron density (ligand not included in the density calculations). Green mesh represents fo-fc omit map difference density for Chain A. Hydrogen bonds to the ligand are shown as dashed black lines with distances indicated in Angstroms (Å). G. The ligand-binding site of serine-bound EcTsr is shown as in F, with omit map fo-fc electron density. The two chains of EcTsr in the AU are overlaid (orange) with one chain of serine-bound SeTsr (blue). H-I. Closeup view of the L-serine ligand and fo-fc omit map density for the SeTsr (blue) and EcTsr (orange) structures, respectively. J-L. Isothermal titration calorimetry analyses of the SeTsr LBD with L-serine, NE, or DHMA. M. Sequence conservation among Enterobacteriaceae with residues of the L-serine ligand-binding pocket highlighted. N. Biological distribution of Tsr homologues. See also Move S7, Data S1, and Table S2. (gray, n=16). Data are means and error is SEM. See also Fig. S2, Fig. S3, Fig. S4, Movie S4, Movie S5, Movie S6.

Enterobacteriaceae possessing Tsr display chemoattraction to human serum. A-C. Response of C. koseri BAA-895 (n=3, 37° C) and E. coli MG1655 to human serum (n=3, 30° C), shown as max projections. Plotted data shown are means averaged over 1 s, and error is SEM. See also Movie S8, Movie S9, and Table S1.

CIRA experimentation controls and microgradient modeling. A. Average injection flow of treatment solution from the glass microcapillary. Flow was measured through injection of known concentrations of methylene blue dye, diluted in CB, into a 50 µl pond of CB over 20 minutes. After treatments, the absorbance of solutions was measured at 665 nm and quantified based on a standard curve. Applying a compensation pressure of 35 hPa resulted in an average flow of 305.5 fl/min ± 31. B. CIRA localization in response to treatments of buffered CB of different pH. C. Comparison of responses of GFP versus mPlum strains using CIRA. D-F. Model of the CIRA microgradient for effectors relevant to this study. Topology maps of 1 mm x 1 mm size are shown for each microgradient in an ‘integrated’ format, which models what is seen by eye from the bottom-up view of the microscopy plane (left), or as the local concentration that would be experienced by a bacterium at a given distance (center). A plane through the center of the treatment sphere is shown (right), with relative concentrations experienced at a given distance expressed in nM units (scale bar is 100 µm).

Comparison of chemotactic responses to serum, serum with serine racemase treatment, L-serine versus DHMA and NE. Shown are max projections from CIRA experiments over a 10 s time period following 300 s of treatments as shown. Experiments F-G utilized cells that were primed with 5 µM NE for 3 hours prior to experimentation.

Calculation of L-serine source required for half-maximal chemoattraction response (K1/2). Data are mean AUC values of relative number of bacteria within 150 µm of the treatment location for the -15 s – 250 s range, represented as fold-change. Data are fit with an exponential curve (red line):

Where a=2.636193, b =-1.616338, and c=0.006282888. K1/2 is approximated to be 105 µm (x, dashed lines).

Total serine present in human serum samples, as determined by mass spectrometry. Treatment with 50 µg recombinant serine dehydratase (SDS) over 4 h did not decrease L-serine content in human serum.

Bacterial strains and plasmids used in this study.

Summary of crystallographic statistics