CryoEM structures reveal how the bacterial flagellum rotates and switches direction.

Bacterial chemotaxis requires bidirectional flagellar rotation at different rates. Rotation is driven by a flagellar motor, which is a supercomplex containing multiple rings. Architectural uncertainty regarding the cytoplasmic C-ring, or 'switch', limits our understanding of how the motor transmits torque and direction to the flagellar rod. Here we report cryogenic electron microscopy structures for Salmonella enterica serovar typhimurium inner membrane MS-ring and C-ring in a counterclockwise pose (4.0 Å) and isolated C-ring in a clockwise pose alone (4.6 Å) and bound to a regulator (5.9 Å). Conformational differences between rotational poses include a 180° shift in FliF/FliG domains that rotates the outward-facing MotA/B binding site to inward facing. The regulator has specificity for the clockwise pose by bridging elements unique to this conformation. We used these structures to propose how the switch reverses rotation and transmits torque to the flagellum, which advances the understanding of bacterial chemotaxis and bidirectional motor rotation.

Structural basis of directional switching by the bacterial flagellum.

The bacterial flagellum is a macromolecular protein complex that harvests energy from uni-directional ion flow across the inner membrane to power bacterial swimming via rotation of the flagellar filament. Rotation is bi-directional, with binding of a cytoplasmic chemotactic response regulator controlling reversal, though the structural and mechanistic bases for rotational switching are not well understood. Here we present cryoelectron microscopy structures of intact Salmonella flagellar basal bodies (3.2-5.5 Å), including the cytoplasmic C-ring complexes required for power transmission, in both counter-clockwise and clockwise rotational conformations. These reveal 180° movements of both the N- and C-terminal domains of the FliG protein, which, when combined with a high-resolution cryoelectron microscopy structure of the MotA5B2 stator, show that the stator shifts from the outside to the inside of the C-ring. This enables rotational switching and reveals how uni-directional ion flow across the inner membrane is used to accomplish bi-directional rotation of the flagellum.

Enhanced sensitivity and accuracy via gold nanoparticles based multi-line lateral flow immunoassay strip for Salmonella typhimurium detection in milk and orange juice.

Food borne pathogens threaten food safety and affect human health. The lateral flow immunoassays (LFIAs) are widely concerned because of simplicity, low cost and user friendliness, and have broad application prospects in pathogen detection. However, the sensitivity of LFIAs is limited. Herein, multi-line LFIAs are introduced into pathogen detection for the first time. Compared with traditional single-line LFIAs, the overall signal strength of multi-line LFIAs has been significantly improved. It is particularly noteworthy that multi-line LFIAs detection accuracy of 103 CFU/mL pathogen has been improved by about 55%. The proposed multi-line LFIAs reduce the possibility of judging a positive result as a false negative result. The LFIAs strip was validated in real samples of milk and orange juice. This strategy has great potential for rapid detection of pathogens in real samples, and provides new insights for improving the accuracy and sensitivity of LFIAs strips.

MntP and YiiP Contribute to Manganese Efflux in Salmonella enterica Serovar Typhimurium under Conditions of Manganese Overload and Nitrosative Stress.

The divalent transition metal cation manganese is important for protein function, particularly under conditions of iron limitation, nitrosative stress, and oxidative stress, but can mediate substantial toxicity in excess. Salmonella enterica serovar Typhimurium possesses multiple manganese importers, but the pathways for manganese efflux remain poorly defined. The S. Typhimurium ATCC 14028s genome was analyzed for putative manganese export pathways, which identified a previously uncharacterized homologue of the Escherichia coli manganese exporter mntP, stm1834, and two cation diffusion facilitator family transporters, zitB (stm0758) and yiiP (stm4061). Manganese acquisition by S. Typhimurium has been shown to occur in response to nitric oxide, an important chemical mediator of the mammalian innate immune response. However, cellular manganese can rapidly return to prechallenge levels, strongly suggesting that one or more S. Typhimurium exporters may contribute to this process. Here, we report that mntP and yiiP contribute to manganese resistance and export in S. Typhimurium. YiiP, also known as FieF, has previously been associated with zinc and iron transport, although its physiological role remains ambiguous due to a lack of zinc-sensitive phenotypes in yiiP mutant strains of S. Typhimurium and E. coli. We report that S. Typhimurium ΔmntP ΔyiiP mutants are exquisitely sensitive to manganese and show that both YiiP and MntP contribute to manganese efflux following nitric oxide exposure. IMPORTANCE Transition metal cations are required for the function of many proteins but can mediate toxicity when present in excess. Identifying transporters that facilitate metal ion export, the conditions under which they are expressed, and the role they play in bacterial physiology is an evolving area of interest for environmental and pathogenic organisms. Determining the native targets of metal transporters has proved challenging since bioinformatic predictions, in vitro transport data, and mutant phenotypes do not always agree. This work identifies two transporters that mediate manganese efflux from the Gram-negative pathogen Salmonella enterica serovar Typhimurium in response to manganese overload and nitric oxide stress. While homologues of MntP have been characterized previously, this is the first observation of YiiP contributing to manganese export.

Molecular basis of glycyl-tRNAGly acetylation by TacT from Salmonella Typhimurium.

Bacterial toxin-antitoxin modules contribute to the stress adaptation, persistence, and dormancy of bacteria for survival under environmental stresses and are involved in bacterial pathogenesis. In Salmonella Typhimurium, the Gcn5-related N-acetyltransferase toxin TacT reportedly acetylates the α-amino groups of the aminoacyl moieties of several aminoacyl-tRNAs, inhibits protein synthesis, and promotes persister formation during the infection of macrophages. Here, we show that TacT exclusively acetylates Gly-tRNAGlyin vivo and in vitro. The crystal structure of the TacT:acetyl-Gly-tRNAGly complex and the biochemical analysis reveal that TacT specifically recognizes the discriminator U73 and G71 in tRNAGly, a combination that is only found in tRNAGly isoacceptors, and discriminates tRNAGly from other tRNA species. Thus, TacT is a Gly-tRNAGly-specific acetyltransferase toxin. The molecular basis of the specific aminoacyl-tRNA acetylation by TacT provides advanced information for the design of drugs targeting Salmonella.

A Selective Tether Recruits Activated Response Regulator CheB to Its Chemoreceptor Substrate.

Methylesterase/deamidase CheB is a key component of bacterial chemotaxis systems. It is also a prominent example of a two-component response regulator in which the effector domain is an enzyme. Like other response regulators, CheB is activated by phosphorylation of an aspartyl residue in its regulatory domain, creating an open conformation between its two domains. Studies of CheB in Escherichia coli and related organisms have shown that its enzymatic action is also enhanced by a pentapeptide-binding site for the enzyme at the chemoreceptor carboxyl terminus. Related carboxyl-terminal pentapeptides are found on >25,000 chemoreceptor sequences distributed across 11 bacterial phyla and many bacterial species, in which they presumably play similar roles. Yet, little is known about the interrelationship of CheB phosphorylation, pentapeptide binding, and interactions with its substrate methylesters and amides on the body of the chemoreceptor. We investigated by characterizing the binding kinetics of CheB to Nanodisc-inserted chemoreceptor dimers. The resulting kinetic and thermodynamic constants revealed a synergy between CheB phosphorylation and pentapeptide binding in which a phosphorylation mimic enhanced pentapeptide binding, and the pentapeptide served not only as a high-affinity tether for CheB but also selected the activated conformation of the enzyme. The basis of this selection was revealed by molecular modeling that predicted a pentapeptide-binding site on CheB which existed only in the open, activated enzyme. Recruitment of activated enzyme by selective tethering represents a previously unappreciated strategy for regulating response regulator action, one that may well occur in other two-component systems. IMPORTANCE Two-component signal transduction systems are a primary means by which bacteria sense and respond to their environment. Response regulators are key components of these systems. Phosphorylation of response regulators by cognate histidine kinases generate active conformations which act on specific targets, DNA sequences or proteins. The targets have been considered passive in this process. Our characterization of interaction between response regulator CheB and its target chemoreceptor revealed active participation of the target in response regulator action. We found that a pentapeptide sequence at the carboxyl terminus of Escherichia coli chemoreceptors is a selective tether that binds only phosphorylated CheB, thus selecting the form of this two-component enzyme active for covalent modification of the selecting chemoreceptor. Analogous pentapeptides are found on chemoreceptors in many bacterial species and are presumably also selective tethers. There may well be other, uncharacterized examples of active participation of target molecules in response to regulator action.

Holistic Characterization of a Salmonella Typhimurium Infection Model Using Integrated Molecular Imaging.

A more complete and holistic view on host-microbe interactions is needed to understand the physiological and cellular barriers that affect the efficacy of drug treatments and allow the discovery and development of new therapeutics. Here, we developed a multimodal imaging approach combining histopathology with mass spectrometry imaging (MSI) and same section imaging mass cytometry (IMC) to study the effects of Salmonella Typhimurium infection in the liver of a mouse model using the S. Typhimurium strains SL3261 and SL1344. This approach enables correlation of tissue morphology and specific cell phenotypes with molecular images of tissue metabolism. IMC revealed a marked increase in immune cell markers and localization in immune aggregates in infected tissues. A correlative computational method (network analysis) was deployed to find metabolic features associated with infection and revealed metabolic clusters of acetyl carnitines, as well as phosphatidylcholine and phosphatidylethanolamine plasmalogen species, which could be associated with pro-inflammatory immune cell types. By developing an IMC marker for the detection of Salmonella LPS, we were further able to identify and characterize those cell types which contained S. Typhimurium.

Genetic Analysis of the Salmonella FliE Protein That Forms the Base of the Flagellar Axial Structure.

The FliE component of the bacterial flagellum is the first protein secreted through the flagellar type III secretion system (fT3SS) that is capable of self-assembly into the growing bacterial organelle. The FliE protein plays dual roles in the assembly of the Salmonella flagellum as the final component of the flagellar type III secretion system (fT3SS) and as an adaptor protein that anchors the rod (drive shaft) of the flagellar motor to the membrane-imbedded MS-ring structure. This work has identified the interactions between FliE and other proteins at the inner membrane base of the flagellar machine. The fliE sequence coding for the 104-amino-acid protein was subject to saturating mutagenesis. Single-amino-acid substitutions were generated in fliE, resulting in motility phenotypes. From these mutants, intergenic suppressor mutations were generated, isolated, and characterized. Single-amino-acid mutations defective in FliE function were localized to the N- and C-terminal helices of the protein. Motile suppressors of amino acid mutations in fliE were found in rod protein genes flgB and flgC, the MS ring gene, fliF, and one of the core T3SS genes, fliR. These results support the hypothesis that FliE acts as a linker protein consisting of an N-terminal α-helix that is involved in the interaction with the MS ring with a rotational symmetry and a C-terminal coiled coil that interacts with FliF, FliR, FlgB, and FlgC, and these interactions open the exit gate of the protein export channel of the fT3SS. IMPORTANCE The bacterial flagellum represents one of biology's most complex molecular machines. Its rotary motor spins at speeds of more than 2,000 cycles per second, and its type 3 secretion (T3S) system secretes proteins at rates of tens of thousands of amino acids per second. Within the complex flagellar motility machine resides a unique protein, FliE, which serves as an adaptor to connect a planar, inner membrane-embedded ring structure, the MS-ring, the core T3S secretion complex at the cytoplasmic base, and a rigid, axial structure that spans the periplasmic space, penetrates the outer membrane, and extends 10 to 20 microns from the cell surface. This work combines genetic mutant suppressor analysis with the structural data for the core T3S system, the MS-ring, and the axial drive shaft (rod) that transverses the periplasm to provide insight into the essential adaptor role of FliE in flagellum assembly and function.

Cellular Activity of Salmonella Typhimurium ArtAB Toxin and Its Receptor-Binding Subunit.

Salmonellosis is among the most reported foodborne illnesses in the United States. The Salmonellaenterica Typhimurium DT104 phage type, which is associated with multidrug-resistant disease in humans and animals, possesses an ADP-ribosylating toxin called ArtAB. Full-length artAB has been found on a number of broad-host-range non-typhoidal Salmonella species and serovars. ArtAB is also homologous to many AB5 toxins from diverse Gram-negative pathogens, including cholera toxin (CT) and pertussis toxin (PT), and may be involved in Salmonella pathogenesis, however, in vitro cellular toxicity of ArtAB has not been characterized. artAB was cloned into E. coli and initially isolated using a histidine tag (ArtABHIS) and nickel chromatography. ArtABHIS was found to bind to African green monkey kidney epithelial (Vero) cells using confocal microscopy and to interact with glycans present on fetuin and monosialotetrahexosylganglioside (GM1) using ELISA. Untagged, or native, holotoxin (ArtAB), and the pentameric receptor-binding subunit (ArtB) were purified from E. coli using fetuin and d-galactose affinity chromatography. ArtAB and ArtB metabolic and cytotoxic activities were determined using Vero and Chinese hamster ovary (CHO) epithelial cells. Vero cells were more sensitive to ArtAB, however, incubation with both cell types revealed only partial cytotoxicity over 72 h, similar to that induced by CT. ArtAB induced a distinctive clustering phenotype on CHO cells over 72 h, similar to PT, and an elongated phenotype on Vero cells, similar to CT. The ArtB binding subunit alone also had a cytotoxic effect on CHO cells and induced morphological rounding. Results indicate that this toxin induces distinctive cellular outcomes. Continued biological characterization of ArtAB will advance efforts to prevent disease caused by non-typhoidal Salmonella.

Molecular Basis for Environment Sensing by a Nucleoid-Structuring Bacterial Protein Filament.

The histone-like nucleoid structuring (H-NS) protein controls the expression of hundreds of genes in Gram-positive bacteria through its capability to coat and condense DNA. This mechanism requires the formation of superhelical H-NS protein filaments that are sensitive to temperature and salinity, allowing H-NS to act as an environment sensor. We use multiscale modeling and simulations to obtain detailed insights into the mechanism of H-NS filament's sensitivity to environmental changes. Through the simulations of the superhelical H-NS filament, we reveal how different environments induce heterogeneity of H-NS monomers. Further, we observe that transient self-association within the H-NS filament creates temperature-inducible strain and might mildly oppose DNA binding. We also probe different H-NS-DNA complex architectures and show that complexation enhances the stability of both DNA and H-NS superhelices. Overall, our results provide unprecedented molecular insights into the environmental sensing and DNA interactions of a prototypical nucleoid-structuring bacterial protein filament.

NMR reveals the interplay between SilE and SilB model peptides in the context of silver resistance.

SilE and SilB are both proteins involved in the silver efflux pump found in Gram-negative bacteria such as S. typhimurium. Using model peptides along with NMR and CD experiments, we show how SilE may store silver ions prior to delivery and we hypothesize for the first time the interplay between SilB and SilE.

Protective Effect of Nigella sativa and Nigella damascena Fixed Oils Against Aflatoxin Induced Mutagenicity in the Classical and Modified Ames Test.

The antioxidant and mutagenic/antimutagenic activities of the fixed oils from Nigella sativa (NSO) and Nigella damascena (NDO) seeds, obtained by cold press-extraction from the cultivar samples, were comparatively investigated for the first time. The antimutagenicity test was carried out using classical and modified Ames tests. The fatty acid composition of the fixed oils was characterized by gas chromatography-mass spectrometry (GC-MS) while the quantification of thymoquinone in the fixed oils was determined by UPC2 . The main components of the NSO and NDO were found to be linoleic acid, oleic acid, and palmitic acid. The results of the Ames test confirmed the safety of NSO and NDO from the viewpoint of mutagenicity. The results of the three antioxidant test methods were correlated with each other, indicating NDO as having a superior antioxidant activity, when compared to the NSO. Both NSO and NDO exhibited a significant protective effect against the mutagenicity induced by aflatoxin B1 in Salmonella typhimurium TA98 and TA100 strains. When microsomal metabolism was terminated after metabolic activation of the mycotoxin, a significant increase in antimutagenic activity was observed, suggesting that the degradation of aflatoxin B1 epoxides by these oils may be a possible antimutagenic mechanism. It is worthy to note that this is the first study to assess the mutagenicity of NSO and NDO according to the OECD 471 guideline and to investigate antimutagenicity of NDO in comparison to NSO against aflatoxin.

OmpC, a novel factor H-binding surface protein, is dispensable for the adherence and virulence of Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium utilizes a series of strategies to evade host innate immune defenses, including the serum complement system. Many microbial pathogens have evolved the ability to bind the complement regulatory protein factor H (FH) through their surface factor H-binding proteins (FHBPs) to circumvent the complement-mediated bactericidal effect. However, the roles of FHBPs in Salmonella pathogenesis are not well understood. In this study, we demonstrated that the survival of S. Typhimurium in human serum was decreased in a time and concentration dependent manner. Pre-incubation with FH attenuated the sensitivity of S. Typhimurium strain χ3761 to complement-mediated serum killing, suggesting FH binding enhance survival in serum. We aimed to identify novel S. Typhimurium FHBPs and characterize their biological functions. Here, six potential FHBPs were identified by two-dimensional (2D)-Far-western blot, and three of them were further confirmed to bind FH by Far-western blot and dot blot. We found that deletion of ompC (ΔompC) significantly inhibited the survival of S. Typhimurium strain χ3761 in human serum. Our results indicated that the ompC mutation does not affect χ3761 adhesion to HeLa cells. Furthermore, a mice infection model showed that deletion of ompC had no significant effect on the histopathological lesions or viability compared with the wild-type strain χ3761. In summary, these results suggested that OmpC is an important FHBP, but not a critical virulence factor of S. Typhimurium.

Dual-mode aptasensor for simultaneous detection of multiple food-borne pathogenic bacteria based on colorimetry and microfluidic chip using stir bar sorptive extraction.

A dual-mode aptasensor using colorimetry and microfluidic chip (MC) together with stir bar sorptive extraction (SBSE) has been developed for firstly qualifying samples contaminated with Vibrio parahaemolyticus (V.P) and Salmonella typhimurium (S.T), then precisely determine both of them in positive samples. For this purpose, the aptamer-streptavidin encoded probes (Apt-SAEs) corresponding to different bacteria were prepared in advance. Then, a stir bar modified with 4-mercaptophenylboronic acid (MPBA) was made to extract bacteria together with Apt-SAE probes. The binding event of aptamer and target triggered the formation of two sandwich structures containing Apt-SAE, V.P or S.T. The concentration of bacteria could be enriched by 1000 times within 15 min to avoid long-time enrichment process. Finally, the stir bar was immersed in the 3,3',5,5'-Tetramethylbenzidine (TMB)-H2O2 solution for color development. The color could be observed by naked eyes to judge whether the analytes were present. The colorless samples were judged to be negative. For the positive samples, the adsorbed encoded probes corresponding to different bacteria would be eluted from the stir bar and rapidly analyzed by the MC. Under the optimized conditions, 100 CFU/mL of V.P or S.T or both of them could be observed by colorimetry and 35 CFU/mL of them could be detected (S/N = 3) by the MC. The assay has significant application value for on-site screening and multiple detection of food-borne pathogenic bacteria.

Structure of the molecular bushing of the bacterial flagellar motor.

The basal body of the bacterial flagellum is a rotary motor that consists of several rings (C, MS and LP) and a rod. The LP ring acts as a bushing supporting the distal rod for its rapid and stable rotation without much friction. Here, we use electron cryomicroscopy to describe the LP ring structure around the rod, at 3.5 Å resolution, from Salmonella Typhimurium. The structure shows 26-fold rotational symmetry and intricate intersubunit interactions of each subunit with up to six partners, which explains the structural stability. The inner surface is charged both positively and negatively. Positive charges on the P ring (the part of the LP ring that is embedded within the peptidoglycan layer) presumably play important roles in its initial assembly around the rod with a negatively charged surface.

Fabrication of gold/silver nanodimer SERS probes for the simultaneous detection of Salmonella typhimurium and Staphylococcus aureus.

Salmonella typhimurium (S. typhimurium) and Staphylococcus aureus (S. aureus) are the two most important foodborne pathogens which can easily cause disease infections. Here, the aptamer-facilitated gold/silver nanodimer SERS probes were built for the simultaneous detection of the two bacteria with the help of magnetic separation enrichment. First, two nanodimer SERS signal probes and two magnetic capture probes each connected with the specific aptamer were fabricated. The distance between gold and silver nanoparticles in the dimer can amplify the Raman signal (Cy3 and Rox) at the junction but modified in the aptamer sequence. Then, after the addition of S. typhimurium and S. aureus, the sandwich-like composite structures "SERS signal probes-target-magnetic capture probes" formed because of the high affinity between aptamer sequences and their target bacteria. Under the optimal experimental conditions, the linear correlations between Raman intensity and the logarithm of the concentration of bacteria were y = 876.95x-67.84 (R2 = 0.9865) for S. typhimurium and y = 1280.43x-1752.6 (R2 = 0.9883) for S. aureus. The SERS detection showed the nanodimer probe had high selectivity. Besides, the recovery experiment in milk sample indicated good accuracy compared with the traditional plate counting method.

Gasdermin E permits interleukin-1 beta release in distinct sublytic and pyroptotic phases.

Cellular inflammasome activation causes caspase-1 cleavage of the pore-forming protein gasdermin D (GSDMD) with subsequent pyroptotic cell death and cytokine release. Here, we clarify the ambiguous role of the related family member gasdermin E (GSDME) in this process. Inflammasome stimulation in GSDMD-deficient cells led to apoptotic caspase cleavage of GSDME. Endogenous GSDME activation permitted sublytic, continuous interleukin-1ß (IL-1ß) release and membrane leakage, even in GSDMD-sufficient cells, whereas ectopic expression led to pyroptosis with GSDME oligomerization and complete liberation of IL-1ß akin to GSDMD pyroptosis. We find that NLRP3 and NLRP1 inflammasomes ultimately rely concurrently on both gasdermins for IL-1ß processing and release separately from their ability to induce cell lysis. Our study thus identifies GSDME as a conduit for IL-1ß release independent of its ability to cause cell death.

Chemogenetic Tags with Probe Exchange for Live-Cell Fluorescence Microscopy.

Fluorogenic protein tagging systems have been less developed for prokaryotes than for eukaryotic cell systems. Here, we extend the concept of noncovalent fluorogenic protein tags in bacteria by introducing transcription factor-based tags, namely, LmrR and RamR, for probe binding and fluorescence readout under aerobic and anaerobic conditions. We developed two chemogenetic protein tags that impart fluorogenicity and a longer fluorescence lifetime to reversibly bound organic fluorophores, hence the name Chemogenetic Tags with Probe Exchange (CTPEs). We present an extensive characterization of 30 fluorophores reversibly interacting with the two different CTPEs and conclude that aromatic planar structures bind with high specificity to the hydrophobic pockets of these tags. The reversible binding of organic fluorophores to the CTPEs and the superior photophysical properties of organic fluorophores enable long-term fluorescence microscopy of living bacterial cells. Our protein tags provide a general tool for investigating (sub)cellular protein localization and dynamics, protein-protein interactions, and prolonged live-cell microscopy, even under oxygen-free conditions.

Structure of the cytoplasmic domain of SctV (SsaV) from the Salmonella SPI-2 injectisome and implications for a pH sensing mechanism.

Bacterial type III secretion systems assemble the axial structures of both injectisomes and flagella. Injectisome type III secretion systems subsequently secrete effector proteins through their hollow needle into a host, requiring co-ordination. In the Salmonella enterica serovar Typhimurium SPI-2 injectisome, this switch is triggered by sensing the neutral pH of the host cytoplasm. Central to specificity switching is a nonameric SctV protein with an N-terminal transmembrane domain and a toroidal C-terminal cytoplasmic domain. A 'gatekeeper' complex interacts with the SctV cytoplasmic domain in a pH dependent manner, facilitating translocon secretion while repressing effector secretion through a poorly understood mechanism. To better understand the role of SctV in SPI-2 translocon-effector specificity switching, we purified full-length SctV and determined its toroidal cytoplasmic region's structure using cryo-EM. Structural comparisons and molecular dynamics simulations revealed that the cytoplasmic torus is stabilized by its core subdomain 3, about which subdomains 2 and 4 hinge, varying the flexible outside cleft implicated in gatekeeper and substrate binding. In light of patterns of surface conservation, deprotonation, and structural motion, the location of previously identified critical residues suggest that gatekeeper binds a cleft buried between neighboring subdomain 4s. Simulations suggest that a local pH change from 5 to 7.2 stabilizes the subdomain 3 hinge and narrows the central aperture of the nonameric torus. Our results are consistent with a model of local pH sensing at SctV, where pH-dependent dynamics of SctV cytoplasmic domain affect binding of gatekeeper complex.

Outer Membrane Structural Defects in Salmonella enterica Serovar Typhimurium Affect Neutrophil Chemokinesis but Not Chemotaxis.

Neutrophils, the first line of defense against pathogens, are critical in the host response to acute and chronic infections. In Gram-negative pathogens, the bacterial outer membrane (OM) is a key mediator of pathogen detection; nonetheless, the effects of variations in its molecular structure on the neutrophil migratory response to bacteria remain largely unknown. Here, we developed a quantitative microfluidic assay that precludes physical contact between bacteria and neutrophils while maintaining chemical communication, thus allowing investigation of both transient and steady-state responses of neutrophils to a library of Salmonella enterica serovar Typhimurium OM-related mutants at single-cell resolution. Using single-cell quantitative metrics, we found that transient neutrophil chemokinesis is highly gradated based upon OM structure, while transient and steady-state chemotaxis responses differ little between mutants. Based on our finding of a lack of correlation between chemokinesis and chemotaxis, we define "stimulation score" as a metric that comprehensively describes the neutrophil response to pathogens. Complemented with a killing assay, our results provide insight into how OM modifications affect neutrophil recruitment and pathogen survival. Altogether, our platform enables the discovery of transient and steady-state migratory responses and provides a new path for quantitative interrogation of cell decision-making processes in a variety of host-pathogen interactions.IMPORTANCE Our findings provide insights into the previously unexplored effects of Salmonella envelope defects on fundamental innate immune cell behavior, which advance the knowledge in pathogen-host cell biology and potentially inspire the rational design of attenuated strains for vaccines or immunotherapeutic strains for cancer therapy. Furthermore, the microfluidic assay platform and analytical tools reported herein enable high-throughput, sensitive, and quantitative screening of microbial strains' immunogenicity in vitro This approach could be particularly beneficial for rapid in vitro screening of engineered microbial strains (e.g., vaccine candidates) as the quantitative ranking of the overall strength of the neutrophil response, reported by "stimulation score," agrees with in vivo cytokine response trends reported in the literature.