Biology subjects

Waldor, M. K.

Publications and source records attributed to Waldor, M. K..

At least 19 recordsLinked to original sources

bioRxiv2026

Transcriptomic timeseries links hepatic gene expression to an early and self-limited systemic response to enteric infection

The liver receives microbe and host signals from the intestine via the portal vein, thereby connecting the gut to systemic physiology. Homeostatic control of the timing of systemic responses is critical to prevent the expansion and dissemination of gut microbes and to mitigate untoward effects from prolonged systemic inflammation, however these mechanisms remain enigmatic. Here, to determine the role of the liver in coordinating systemic immune responses to enteric infection, matched measurements of global gene expression profiles were collected from the murine liver and intestinal epithelium throughout the course of enteric infection and clearance of Citrobacter rodentium, a mouse model of infectious colitis. These data revealed metabolic suppression in the liver during the peak of infection and a long-lived immune signaling pattern in the colon associated with CD4 and CD8 T cell infiltration that persisted beyond the clearance of infection. Furthermore, an early inflammatory signal was detected in the liver that resolved before the peak of disease and pathogen colonization. This self-limited, early signal depended on the pathogens virulence program and correlated with the timing of a corresponding systemic response, including circulating TNF- and IL-6, key mediators of acute-phase proteins. These results uncover the temporal pattern of hepatic changes in response to the course of intestinal infection and provide correlative evidence that an early pulse of gene expression in the liver coordinates and limits the duration of the systemic acute-phase protein response. Author summaryLocalized infections can trigger systemic inflammatory responses that help limit infection. However, prolonged systemic inflammation risks tissue damage and disease. Thus, the timing of systemic immune reactions is critical to the balance between immunity and damage. Yet, knowledge of the pathways that link gut-localized infections to systemic immune tone is limited. Since the liver is anatomically central to the connection between the gut and circulatory systems, we hypothesized that monitoring the kinetics of liver gene expression throughout the natural course of an intestinal bacterial infection would provide a valuable resource and yield insight into the control of systemic immune tone. Critically, we identified a burst of liver cytokine signaling that occurred and resolved before peak pathogen burden and disease in the colon and predicted the circulating inflammatory response. We propose that this early and self-limited signal from the liver coordinates the timing of the systemic response, ensuring it occurs early enough to promote immunity and resolve before causing tissue damage.

immunology↗

bioRxiv2025

Vibrio cholerae serotype impacts pathogenicity

The O1 serogroup of Vibrio cholerae has caused all cholera pandemics and for over a century V. cholerae O1 outbreak strains have been characterized by their serotype. The two V. cholerae serotypes differ by the presence (Ogawa) or absence (Inaba) of methylation of the terminal sugar on the lipopolysaccharide O1-antigen. Serotype switching often occurs during epidemics and has historically been attributed to the pathogen adapting to immune pressures. Here we address the impact of serotype on V. cholerae pathogenicity using otherwise isogenic Ogawa and Inaba versions of several clinical V. cholerae O1 isolates. Our findings indicate that O1 antigen methylation in Ogawa strains promotes V. cholerae colonization, infectivity and resistance to antimicrobial peptides. We propose that methylation of the O1 antigen elevates colonization by shielding the bacterium from cationic antimicrobial peptides at the pH of the small intestine. These observations provide insights into the biological significance of the V. cholerae O1 serotypes.

microbiology↗

bioRxiv2025

Adapting CRISPR-associated transposons for rapid and high-throughput reverse genetics

CRISPR-associated transposons (CAST) use guide RNAs to direct their transposition and are being harnessed as tools for programmable genome engineering across diverse bacterial species. However, CAST systems have not been adapted for high-throughput genetic screening. Here, we present MultiCAST, a streamlined platform for rapid and scalable guide RNA-directed transposon insertion in bacteria. MultiCAST generates targeted insertions in a single step through conjugative delivery of conditionally replicative plasmids encoding the CAST enzymatic machinery and a selectable mini-transposon expressing the guide RNA. By leveraging the inserted guide sequence as a molecular barcode, MultiCAST enables pooled, high-throughput genetic screens using only amplicon sequencing. We identified factors that influence transposition efficiency and the accuracy of insertion frequency measurements derived from guide sequencing. Adjusting the ratio of donor and recipient strain during conjugation mitigates guide-transposon "crosstalk", in which a single recipient cell acquires multiple donor plasmids containing distinct guides. Furthermore, we developed a machine learning-based predictive model for selecting highly active guides based on target sequence features that strongly correlate with activity. The nucleoid-associated protein H-NS was also found to inhibit CAST activity, providing a mechanistic explanation for variable insertion frequencies among non-essential genes. To demonstrate the scalability of MultiCAST, we screened a pooled mutant population created from >5,200 guides targeting 88 genes in E. coli across twelve nutrient conditions, accurately identifying genes with condition-specific fitness effects. The simplicity, speed, and throughput of MultiCAST make genome-scale functional screens more accessible across a wide range of bacterial species. SignificanceEfficient gene disruption is essential for understanding bacterial gene function, but traditional genetic approaches are labor-intensive and generally not well-suited for high-throughput studies. We developed MultiCAST, a simple and scalable method that harnesses guide RNA-directed CRISPR-associated transposons for targeted bacterial gene disruption. MultiCAST enables single and pooled transposon mutagenesis in a single step and eliminates the need for complex sequencing library preparation protocols by using the guide sequence as a quantifiable surrogate for mutant abundance. This approach allows thousands of mutants to be generated and screened simultaneously across multiple conditions using only amplicon sequencing. By dramatically reducing the time, cost, and complexity of reverse genetics, MultiCAST opens new possibilities for genome-scale functional studies, accelerating the discovery of bacterial gene functions.

microbiology↗

bioRxiv2025

IscR-mediated sensing of iron-sulfur cluster demand coordinates virulence gene expression in Yersinia pseudotuberculosis

The type III secretion system (T3SS) is a needle-like appendage that translocates effector proteins into host cells to disrupt host defenses. Strict control of T3SS expression is critical for facultative pathogens such as Yersinia pseudotuberculosis, as the T3SS is indispensable for virulence but is metabolically costly. We previously showed that the iron-sulfur (Fe-S) cluster-coordinating transcription factor IscR controls expression of LcrF, the master regulator of the Yersinia T3SS and the YadA adhesin. Clusterless apo-IscR, the predominant IscR form during high cellular Fe-S cluster demand (aerobic, low iron conditions), promotes LcrF, T3SS, and YadA expression. Importantly, binding of apo-IscR to the lcrF promoter at a type II IscR binding site facilitates Yersinia disseminated infection. Here, we show that mutating the lcrF promoter to allow only [2Fe-2S]-IscR binding (lcrFpTypeI) results in hyperexpression of LcrF, the T3SS, and YadA during low Fe-S cluster demand (anaerobic, iron replete conditions). These data suggest that switching the form of IscR that can bind to the lcrF promoter reversed how iron and oxygen regulate Yersinia virulence factors. We used barcoded Y. pseudotuberculosis to probe how control of the lcrF promoter in response to iron and oxygen modifies infection dynamics. We found that the lcrFpTypeI mutant experiences a tighter bottleneck in the cecum, where Yersinia is expected to experience a low oxygen, iron replete environment. Taken together, these findings suggest that by tying T3SS and YadA expression to cellular Fe-S cluster demand, Yersinia can fine-tune its virulence repertoire to the host tissue microenvironment. ImportanceIron and oxygen availability fluctuate spatially across mammalian tissues as well as temporally during the course of bacterial infection. The [2Fe-2S] cluster coordinating transcription factor IscR senses changes in iron and oxygen levels, and plays a pivotal role in enabling pathogens like Yersinia, Salmonella, and Vibrio to express critical virulence genes. While prior research has established that iron availability and oxygen tension influence IscR abundance and DNA-binding specificity, it is unclear how these changes control the timing and location of virulence factor expression during infection. In this study, we engineered a bacterial strain to reverse the way in which iron and oxygen drive expression of two critical virulence factors through IscR. This mutant displayed altered host infection dynamics, revealing that uncoupling virulence gene expression from host tissue cues decreases bacterial fitness.

microbiology↗

bioRxiv2025

Atlas of innate immune responses to experimental cholera and IL22 treatment demonstrates protection by mucus-secreting cells

The diarrheal disease cholera remains a global threat, but there is limited knowledge of the innate immune defenses in the small intestine that protect against the causative agent, Vibrio cholerae. Here, single-cell RNA-sequencing of epithelial and immune cells mapped gene expression patterns in the infant mouse small intestine and revealed changes in response to V. cholerae infection and prophylactic treatment with an IL22 Fc-fusion protein. Infection increased the abundance of an enterocyte subtype with high expression of defense-associated functions and stimulated production of IL22, a cytokine linked to epithelial integrity, from group 3 innate lymphoid cells. Administration of IL22Fc increased production of vibriocidal Reg3{beta} from enterocytes and the abundance of secretory lineage and Muc2-producing goblet cells, which secreted mucus into the intestinal crypts, impairing V. cholerae association with the epithelium. These IL22-mediated responses limited V. cholerae intestinal colonization and protected mice from diarrhea and death. Our findings suggest enterocyte specialization in mucosal defense. SummarySingle cell studies uncovered specialization of epithelial cells in intestinal defense. The epithelial cell-targeting cytokine IL22 protected mice from cholera by promoting the genesis of mucus secreting cells.

microbiology↗

bioRxiv2025

Anoxia activates CRISPR-Cas immunity in the intestine

The natural context in which CRISPR-Cas systems are active in Enterobacteriaceae has remained enigmatic. Here, we find that the Citrobacter rodentium Type I-E CRISPR-Cas system is activated by the oxygen-responsive transcriptional regulator Fnr in the anoxic mouse intestine. Since Fnr-dependent regulation is predicted in ~41% of Enterobacteriaceae cas3 orthologs, we propose that anoxic regulation of CRISPR-Cas immunity is an adaptation that protects Enterobacteriaceae against threats arising from the intestinal microbiome.

microbiology↗

bioRxiv2025

A connection between Vibrio cholerae motility and inter-animal transmission

Outbreaks of cholera are caused by the highly transmissive pathogen Vibrio cholerae. Here, a transposon screen revealed that inactivation of the V. cholerae motility-linked gene motV increases infant mouse intestinal colonization. Compared to wild-type V. cholerae, a {Delta}motV mutant, which exhibits heightened motility in the form of constitutive straight swimming, localizes to the crypts earlier in infection and over a larger area of the small intestine. Aberrant localization of the mutant was associated with an increased number of V. cholerae initiating infection, and elevated pathogen burden, diarrhea, and lethality. Moreover, the deletion of motV causes V. cholerae to transmit from infected suckling mice to naive littermates more efficiently. Even in the absence of cholera toxin, the {Delta}motV mutant continues to transmit between animals, although less than in the presence of toxin, indicating that phenotypes other than cholera toxin-driven diarrhea contribute to transmission. Collectively, this work provides experimental evidence linking intra-animal bottlenecks, colonization, and disease to inter-animal transmission.

microbiology↗

bioRxiv2025

Lethal streptococcal infection is associated with widened lymph node bottlenecks

Streptococcus suis serotype 2 (SS2) infection causes streptococcal toxic shock-like syndrome (STSLS), which can be lethal in both pigs and humans. In mouse models, relatively subtle differences in the inoculum size result in drastically different infection outcomes, from clearance to acute death. Here, we leveraged barcoded SS2 in a murine model to define the bacterial population dynamics associated with death. Barcode sequencing analysis demonstrated that a 10-fold increase in inoculum size led to a 100-1,000-fold increase in the number of bacteria that initiated infection in certain lymph nodes, ultimately driving lethality. This lethal dose threshold was in part driven by suilysin (SLY), the major SS2 virulence factor. At higher doses, the clearance and replication kinetics of bacteria lacking SLY mimicked WT SS2 at sub-lethal doses. Our findings suggest that acute death in SS2 infection is caused by a wholesale failure of the immune system, enabling a large number of bacteria to initiate infection. These results demonstrate how subtle changes in inoculum size can have dramatic consequences for infection outcome and how lethal infection is driven by widened infection bottlenecks. Author SummaryLethal infections are often associated with a specific dose threshold, where increasing inoculum size is associated with an increased likelihood of death. However, a quantitative explanation how this numeric threshold is established is lacking. For example, lethal dose thresholds may reflect a dose where at least one microbe can pass through host-imposed barriers to infection. Alternatively, lethal dose thresholds may reflect a failure of the host immune system, leading to numerous microbes initiating infection. Here, using barcoded Streptococcus suis serotype 2 (SS2), we compare the within-host population dynamics associated between lethal and non-lethal infections. We find that lethal doses are associated with dramatic widening of infection bottlenecks, leading to a large number of bacteria that initiate infection. Lethal infection is also driven by suilysin, the major SS2 virulence factor. These observations demonstrate how lethal doses are quantitatively established by the relationship between host bottlenecks and bacterial virulence factors.

microbiology↗

bioRxiv2024

The Mla pathway promotes Vibrio cholerae re-expansion from stationary phase

Bacteria have evolved diverse strategies to ensure survival under nutrient-limited conditions, where rapid energy generation is not achievable. Here, we performed a transposon insertion site sequencing loss-of-function screen to identify Vibrio cholerae genes that promote the pathogens fitness in stationary phase. We discovered that the Mla (maintenance of lipid asymmetry) pathway, which is crucial for transferring phospholipids from the outer to the inner membrane, is critical for stationary phase fitness. Competition experiments with barcoded and fluorophore labeled wild-type and mlaE mutant V. cholerae revealed that the Mla pathway promotes re-expansion from 48h stationary phase cultures. The mutants defect in transitioning out of stationary phase into active growth (culturability) was also observed in monocultures at 48h. However, by 96h the culturability of the mutant and wild-type strains were equivalent. By monitoring the abundances of genomically barcoded libraries of wild-type and {Delta}mlaE strains, we observed that a few barcodes dominated the mutant culture at 96h, suggesting that the similarity of the population sizes at this time was caused by expansion of a subpopulation containing a mutation that suppressed the mlaE mutants defect. Whole genome sequencing revealed that mlaE suppressors inactivated flagellar biosynthesis. Additional mechanistic studies support the idea that the Mla pathway is critical for the maintenance of V. choleraes culturability as it promotes energy homeostasis, likely due to its role in regulating outer membrane vesicle shedding. Together our findings provide insights into the cellular processes that control re-expansion from stationary phase and demonstrate a previously undiscovered role for the Mla pathway. ImportanceBacteria regularly encounter conditions with nutrient scarcity, where cell growth and division are minimal. Knowledge of the pathways that enable re-growth following nutrient restriction are limited. Here, using the cholera pathogen, we uncovered a role for the Mla pathway, a system that enables phospholipid re-cycling, in promoting Vibrio cholerae re-expansion from stationary phase cultures. Cells labeled with DNA barcodes or fluorophores were useful to demonstrate that though the abundances of wild-type and Mla mutant cells were similar in stationary phase cultures, they had marked differences in their capacities to regrow on plates. Of note, Mla mutant cells lose cell envelope components including high energy phospholipids due to OMV shedding. Our findings suggest that the defects in cellular energy homeostasis which emerge in the absence of the Mla pathway underlie its importance in maintaining V. cholerae culturability.

microbiology↗

bioRxiv2024

Pseudomonas aeruginosa population dynamics in a vancomycin-induced murine model of gastrointestinal carriage

Pseudomonas aeruginosa is a common nosocomial pathogen and a major cause of morbidity and mortality in hospitalized patients. Multiple reports highlight that P. aeruginosa gastrointestinal colonization may precede systemic infections by this pathogen. Gaining a deeper insight into the dynamics of P. aeruginosa gastrointestinal carriage is an essential step in managing gastrointestinal colonization and could contribute to preventing bacterial transmission and progression to systemic infection. Here, we present a clinically relevant mouse model relying on parenteral vancomycin pretreatment and a single orogastric gavage of a controlled dose of P. aeruginosa. Robust carriage was observed with multiple clinical isolates, and carriage persisted for up to 60 days. Histological and microbiological examination of mice indicated that this model indeed represented carriage and not infection. We then used a barcoded P. aeruginosa library along with the sequence tag-based analysis of microbial populations (STAMPR) analytic pipeline to quantify bacterial population dynamics and bottlenecks during the establishment of the gastrointestinal carriage. Analysis indicated that most of the P. aeruginosa population was rapidly eliminated in the stomach, but the few bacteria that moved to the small intestine and the caecum expanded significantly. Hence, the stomach constitutes a significant barrier against gastrointestinal carriage of P. aeruginosa, which may have clinical implications for hospitalized patients. IMPORTANCEWhile P. aeruginosa is rarely part of the normal human microbiome, carriage of the bacterium is quite frequent in hospitalized patients and residents of long-term care facilities. P. aeruginosa carriage is a precursor to infection. Options for treating infections caused by difficult-to-treat P. aeruginosa strains are dwindling, underscoring the urgency to better understand and impede pre-infection stages, such as colonization. Here, we use vancomycin-treated mice to model antibiotic-treated patients who become colonized with P. aeruginosa in their gastrointestinal tracts. We identify the stomach as a major barrier to the establishment of gastrointestinal carriage. These findings suggest that efforts to prevent gastrointestinal colonization should focus not only on judicious use of antibiotics but also on investigation into how the stomach eliminates orally ingested P. aeruginosa.

microbiology↗

bioRxiv2024

Integrating tDNA Epigenomics and Expression with Codon Usage Unravel an Intricate Connection with Protein Expression Dynamics in Trypanosoma cruzi

Codon usage bias impacts protein expression across all kingdoms of life, including trypanosomatids. These protozoa, such as the Trypanosoma cruzi, primarily regulate their protein-coding genes through posttranscriptional mechanisms. Here, we integrated analyses of codon usage with multiple high-throughput sequencing data to investigate whether codon usage bias is present into surface virulence factors (disruptive compartment), conserved housekeeping proteins (core compartment), and proteins involved in the developmental stages of T. cruzi. For the first time in trypanosomatids, tRNA sequencing was employed to reveal coadaptation between codon usage and anticodon availability. Despite notable differences in the proteomes of infective and non-infective forms, they exhibited similar pools of tRNAs and similar codon usage preferences. We observed that open chromatin levels of tRNA genes correlate with tRNA expression in non-infective forms, but not in infective forms, suggesting chromatin states do not control the tRNA pool in the latter. Our analysis also revealed a relationship between anticodon:codon pairing modes and protein abundance. Highly expressed mRNAs favored Watson- Crick base pairing, whereas less expressed mRNAs displayed more wobble base pairing. Overall, our findings suggest that protein expression in T. cruzi is influenced by a combination of codon usage bias, tRNA abundance, and anticodon:codon pairing modes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/602108v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1f63f77org.highwire.dtl.DTLVardef@1c0abf8org.highwire.dtl.DTLVardef@5a4988org.highwire.dtl.DTLVardef@1918f49_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Quantification of Salmonella enterica serovar Typhimurium Population Dynamics in Murine Infection Using a Highly Diverse Barcoded Library

Murine models are often used to study the pathogenicity and dissemination of the enteric pathogen Salmonella enterica serovar Typhimurium. Here, we quantified S. Typhimurium population dynamics in mice using the STAMPR analytic pipeline and a highly diverse S. Typhimurium barcoded library containing [~]55,000 unique strains distinguishable by genomic barcodes by enumerating S. Typhimurium founding populations and deciphering routes of spread in mice. We found that a severe bottleneck allowed only one in a million cells from an oral inoculum to establish a niche in the intestine. Furthermore, we observed compartmentalization of pathogen populations throughout the intestine, with few barcodes shared between intestinal segments and feces. This severe bottleneck widened and compartmentalization was reduced after streptomycin treatment, suggesting the microbiota plays a key role in restricting the pathogens colonization and movement within the intestine. Additionally, there was minimal sharing between the intestine and extraintestinal organ populations, indicating dissemination to extraintestinal sites occurs rapidly, before substantial pathogen expansion in the intestine. Bypassing the intestinal bottleneck by inoculating mice via intravenous or intraperitoneal injection revealed that Salmonella re-enters the intestine after establishing niches in extraintestinal sites by at least two distinct pathways. One pathway results in a diverse intestinal population. The other re-seeding pathway is through the bile, where the pathogen is often clonal, leading to clonal intestinal populations and correlates with gallbladder pathology. Together, these findings deepen our understanding of Salmonella population dynamics. Significance StatementSalmonella is a prevalent food-borne pathogen that infects hundreds of millions of people worldwide. Here, we created a highly complex barcoded Salmonella enterica serovar Typhimurium library containing [~]55,000 barcodes to further understand and quantify Salmonella population dynamics in experimental murine infection. Through comparisons of barcode abundance and frequency in different samples and following different routes of inoculation, we quantify key facets of Salmonella infection, including bottleneck sizes and dissemination patterns, and uncover hidden routes of spread that drive heterogeneity in infection outcome. These observations provide a detailed map of Salmonella infection and demonstrate the power of high-diversity barcoded libraries in deciphering microbial population dynamics.

microbiology↗

bioRxiv2024

Inducible transposon mutagenesis for genome-scale forward genetics

Transposon insertion sequencing (Tn-seq) is a powerful method for genome-scale functional genetics in bacteria. However, its effectiveness is often limited by a lack of mutant diversity, caused by either inefficient transposon delivery or stochastic loss of mutants due to population bottlenecks. Here, we introduce "InducTn-seq", which leverages inducible mutagenesis for temporal control of transposition. InducTn-seq generates millions of transposon mutants from a single colony, enabling the sensitive detection of subtle fitness defects and transforming binary classifications of gene essentiality into a quantitative fitness measurement across both essential and non-essential genes. Using a mouse model of infectious colitis, we show that InducTn-seq bypasses a highly restrictive host bottleneck to generate a diverse transposon mutant population from the few cells that initiate infection, revealing the role of oxygen-related metabolic plasticity in pathogenesis. Overall, InducTn-seq overcomes the limitations of traditional Tn-seq, unlocking new possibilities for genome-scale forward genetic screens in bacteria.

microbiology↗

bioRxiv2024

A pervasive large conjugative plasmid mediates multispecies biofilm formation in the intestinal microbiota increasing resilience to perturbations

Although horizontal gene transfer is pervasive in the intestinal microbiota, we understand only superficially the roles of most exchanged genes and how the mobile repertoire affects community dynamics. Similarly, little is known about the mechanisms underlying the ability of a community to recover after a perturbation. Here, we identified and functionally characterized a large conjugative plasmid that is one of the most frequently transferred elements among Bacteroidales species and is ubiquitous in diverse human populations. This plasmid encodes both an extracellular polysaccharide and fimbriae, which promote the formation of multispecies biofilms in the mammalian gut. We use a hybridization-based approach to visualize biofilms in clarified whole colon tissue with unprecedented 3D spatial resolution. These biofilms increase bacterial survival to common stressors encountered in the gut, increasing strain resiliency, and providing a rationale for the plasmids recent spread and high worldwide prevalence.

microbiology↗

bioRxiv2024

Patterns of Klebsiella pneumoniae bacteremic dissemination from the lung

Bacteremia, a leading cause of death, generally arises after bacteria establish infection in a particular tissue and transit to secondary sites. Studying dissemination from primary sites by solely measuring bacterial burdens does not capture the movement of individual clones. Here, by barcoding Klebsiella pneumoniae, a leading cause of bacteremia, we tracked pathogen dissemination following pneumonia. Variability in organ bacterial burdens was attributable to two distinct dissemination patterns distinguished by the extent of clonal expansion in the lungs. In "metastatic" dissemination, bacterial clones underwent heterogeneous clonal expansion within the lung and the dominant clones spread to secondary organs. In "direct" dissemination, bacterial clones exited the lungs without clonal expansion, leading to lower burdens in systemic sites. We uncover bacterial and host factors that govern these two modes of dissemination. Our data reveal unexpected heterogeneity in the dynamics of Klebsiella bacteremia and define a new paradigm for understanding within-host bacterial dissemination.

microbiology↗

bioRxiv2023

Genetic and immune determinants of E. coli liver abscess formation

Systemic infections can yield distinct outcomes in different tissues. In mice, intravenous inoculation of E. coli leads to bacterial replication within liver abscesses while other organs such as the spleen largely clear the pathogen. Abscesses are macroscopic necrotic regions that comprise the vast majority of the bacterial burden in the animal, yet little is known about the processes underlying their formation. Here, we characterize E. coli liver abscesses and identify host determinants of abscess susceptibility. Spatial transcriptomics revealed that liver abscesses are associated with heterogenous immune cell clusters comprised of macrophages, neutrophils, dendritic cells, innate lymphoid cells, and T-cells that surround necrotic regions of the liver. Susceptibility to liver abscesses is heightened in the C57BL/6 lineage, particularly in C57BL/6N females. Backcross analyses demonstrated that abscess susceptibility is a polygenic trait inherited in a sex-dependent manner without direct linkage to sex chromosomes. As early as one day post infection, the magnitude of E. coli replication in the liver distinguishes abscess-susceptible and abscess-resistant strains of mice, suggesting that the immune pathways that regulate abscess formation are induced within hours. We characterized the early hepatic response with single-cell RNA sequencing and found that mice with reduced activation of early inflammatory responses, such as those lacking the LPS receptor TLR4, are resistant to abscess formation. Experiments with barcoded E. coli revealed that TLR4 mediates a tradeoff between abscess formation and bacterial clearance. Together, our findings define hallmarks of E. coli liver abscess formation and suggest that hyperactivation of the hepatic innate immune response drives liver abscess susceptibility. ImportanceAnimal models of disseminating bacterial infections are critical for developing therapeutic interventions. Following systemic dissemination in mice, E. coli undergo dramatic replication within abscesses in the liver but not in other organs. Although liver abscesses are the largest reservoir of bacteria within the animal, the processes that lead to abscess formation are not known. Here, we characterize E. coli liver abscess formation and identify several determinants of abscess susceptibility, including sex, mouse genotype, and innate immune factors. By combining spatial and single-cell transcriptomics with genetic and phenotypic analyses, we delineate critical host pathways that underlie abscess formation. Our findings define several avenues for future studies to unravel how abscess susceptibility determinants interact to modulate clearance of systemic infections and govern tissue-specific bacterial replication.

microbiology↗

bioRxiv2023

Innate immune responses yield tissue-specific bottlenecks that scale with pathogen dose

To cause infection, pathogens must overcome bottlenecks imposed by the host immune system. These bottlenecks restrict the inoculum and largely determine whether pathogen exposure results in disease. Infection bottlenecks therefore quantify the effectiveness of immune barriers. Here, using a model of Escherichia coli systemic infection, we identify bottlenecks that tighten or widen with higher inoculum sizes, revealing that the efficacy of innate immune responses can increase or decrease with pathogen dose. We term this concept "dose scaling". During E. coli systemic infection, dose scaling is tissue specific, dependent on the LPS receptor TLR4, and can be recapitulated by mimicking high doses with killed bacteria. Scaling is therefore due to sensing of pathogen molecules rather than interactions between the host and live bacteria. We propose that dose scaling quantitatively links innate immunity with infection bottlenecks and is a valuable framework for understanding how the inoculum size governs the outcome of pathogen exposure.

microbiology↗

bioRxiv2023

Peripheral serotonergic neurons regulate anxiety-like behavior and intestinal barrier immunity

Serotonergic circuits in the central nervous system play important roles in regulating mood and behavior, yet the functions of peripheral serotonergic neurons are less understood. Here, we engineered mice lacking the serotonin-producing enzyme Tph2 in peripheral neurons but with intact Tph2 in central neurons. In contrast to mice lacking Tph2 in all neurons, mice lacking Tph2 in peripheral serotonergic neurons did not exhibit increased territorial aggression. However, similar to the total body Tph2 KO mice, the conditional KO animals, exhibited reduced gut motility and decreased anxiety-like behavior. These observations reveal that peripheral serotonergic neurons contribute to control of intestinal motility and anxiety-like behavior and suggest that therapeutics targeting this subset of peripheral neurons could be beneficial.

immunology↗