Extraintestinal pathogenic Escherichia coli (ExPEC) frequently produce Group 2 or 3 capsules that enhance virulence, yet genetic regulation and specific roles of Group 3 capsules remain poorly understood. Here, we demonstrate that the K96 Group 3 capsule in ExPEC strain M12 confers resistance to human serum complement. Using genetic screens, we identified phase-variable mutations in kpsC and essential roles for transcriptional regulators OxyR and RfaH in activating capsule synthesis via a distant promoter. Mutants lacking OxyR or RfaH failed to produce capsule, exhibited complement sensitivity, and showed attenuated virulence in Galleria mellonella. These findings reveal novel mechanisms controlling Group 3 capsule expression and underscore the capsule's contribution to immune evasion.IMPORTANCEGroup 2 capsules are established extraintestinal pathogenic Escherichia coli (ExPEC) virulence factors and are the type most often associated with human isolates. Group 3 capsules have previously been considered a sub-type of Group 2, although nothing was known about factors controlling their expression. Capsule serotype K96-encoding ExPEC strains are increasingly isolated from human infections and inhabit numerous other hosts. It is critical to understand the factors that enable their pathogenic versatility and survival in specific environments. Our study shows that the K96 capsule of strain M12 is required for human complement resistance. Additionally, we have identified genetic factors that control Group 3 capsule synthesis, including a potential phase-variable mechanism as well as transcriptional control by OxyR. Co-regulation of Group 3 capsule synthesis genes with genes necessary for oxidative stress resistance may increase the virulence of some versatile ExPEC strains.

The toxicity of the amino acids glycine and L-serine at high concentrations in bacteria was discovered decades ago. In this work, we used deep transposon insertion sequencing (Tn-seq) experiments to determine the genes necessary to tolerate excess L-serine, diglycine or glycine in the human pathogen Staphylococcus aureus . Our results indicate that intracellular accumulation of specific counterbalancing amino acids-such as alanine in excess glycine-is the primary mechanism of resistance to amino acid toxicity. Consistent with this model, specific amino acid and peptide uptake transporters were required for fitness in each treatment; the peptide transporter DtpT was crucial for fitness in excess L-serine or glycine, and the alanine transporter AapA was essential in diglycine. Tn-seq results also identified the cystine/cysteine uptake transporter TcyABC as necessary in excess L-serine, suggesting that both peptide and cysteine uptake contribute to L-serine tolerance. In addition to uptake mechanisms, glycine and diglycine toxicity is neutralized by D-alanine aminotransferase (Dat), which is required for D-alanine synthesis. The requirement for Dat and DtpT function-but not AapA-in excess glycine is explained by excess glycine inhibiting alanine uptake. Building on this finding, we found that combined treatment with glycine and the alanine analog antibiotic D-cycloserine was strongly synergistic in inhibiting S. aureus growth. Overall, our findings identify targetable mechanisms underlying excess amino acid tolerance in S. aureus , with implications for developing novel combination treatments using the accessible and biocompatible amino acids glycine and L-serine. IMPORTANCE:Growing evidence supports the beneficial effects of glycine and L-serine supplementation in combating bacterial infections. Previous researchers have found that combining antibiotic treatment with high glycine concentrations has additive effects with many antibiotics, even reversing resistance to antibiotics in some bacteria. In vivo , activating glycine and L-serine metabolism heightens the sensitivity of bacterial pathogens to the host complement system, and studies of glycine or L-serine treatments show low toxicity and reduced inflammation in mouse and human subjects. This study reveals that glycine may be an effective antibiotic adjuvant with D-cycloserine, and treatment with glycine or L-serine could potentiate other drugs that target alanine or cysteine metabolism, respectively.

The cell envelope of Gram-positive bacteria is a primary target of host immune defenses and antibiotics, and its stability is influenced by environmental factors, including the availability of the divalent cations Mg2+ and Ca2+. Alanine also plays a critical role in cell envelope integrity, contributing to peptidoglycan cross-linking, D-alanine modification of teichoic acids, and protein synthesis. However, how these factors functionally interact to maintain envelope stability in S. aureus remains unclear. Here, we demonstrate that growth of S. aureus under Mg2+-limited and Ca2+-limited conditions requires increased alanine uptake mediated by the transporter AapA. Loss of AapA results in increased cell lysis and impaired growth under cation-limited conditions, and removing alanine from the growth medium phenocopies these aapA mutant defects. Alanine limitation increases susceptibility to the detergent Triton X-100 and the membrane-targeting antibiotic daptomycin, consistent with defects in envelope stability. Furthermore, aapA function contributes to bacterial fitness in insect and murine infection models. Together, these findings indicate that Mg2+, Ca2+, and alanine play overlapping roles in stabilizing the S. aureus cell envelope, pointing to AapA as a target that may leveraged to enhance antimicrobial efficacy.

Extraintestinal pathogenic Escherichia coli (ExPEC) often produce capsules belonging to Groups 2 or 3, which contribute to invasive disease in humans and other animals. Group 3 capsule loci contain conserved kps genes flanking serotype-specific glycosyltransferase and nucleotide-sugar biosynthesis genes, but little is known about genetic factors that control synthesis, or the specific roles that K96 capsules play in virulence. Previously, we identified a Group 3 serotype K96 capsule in a mastitis-associated strain M12 that is critical for its survival in some host tissues. In this study, we show that the K96 capsule of strain M12 confers resistance to human serum complement. We conducted genetic screens to determine how K96 capsule expression is controlled, which led to two principal findings. First, Group 3 capsule synthesis is unstable. Sequencing of spontaneously appearing mutants revealed potential phase-variable control of capsule expression. Some mutants harbored reversible frameshift mutations in a homopolymeric site within kpsC , which were also identified in other K96-encoding ExPEC isolates. Second, a transposon mutagenesis screen revealed that capsule synthesis requires proteins encoded outside of the kps locus, including the RfaH antiterminator and OxyR transcriptional regulator, which control activation of an unusually distant promoter. An Δ oxyR mutant of strain M12 failed to produce capsule, was extremely sensitive to complement-mediated killing, and avirulent in Galleria mellonella . Importance:Group 2 capsules are established extraintestinal pathogenic Escherichia coli (ExPEC) virulence factors and are the type most often associated with human isolates. Group 3 capsules have previously been considered a sub-type of Group 2, although nothing was known about factors controlling their expression. Capsule serotype K96-encoding ExPEC strains are increasingly isolated from human infections and inhabit numerous other hosts. It is critical to understand the factors that enable their pathogenic versatility and survival in specific environments. Our study shows that K96 capsule of strain M12 is required for human complement resistance. Additionally, we have identified genetic factors that control Group 3 capsule synthesis, including a potential phase-variable mechanism as well as transcriptional control by OxyR. Co-regulation of Group 3 capsule synthesis genes with genes necessary for oxidative stress resistance may increase the virulence of some versatile ExPEC strains.

Gallium is a promising antibacterial candidate because it displaces iron atoms inside bacterial cells but does not undergo redox cycling. It inhibits growth by disrupting essential iron-dependent processes. However, Escherichia coli are naturally less sensitive to gallium than many other bacteria, and the mechanisms that control gallium tolerance are not completely understood. We performed a genome-wide transposon sequencing (TnSeq) screen to identify genes important for the survival of an extraintestinal pathogenic E. coli isolate (M12) in gallium nitrate. The TnSeq results indicated that inactivation of enterobactin siderophore-related genes (entS, fepD, fes, and fepB) enhances bacterial survival in gallium, while disrupting the ferric dicitrate transport system increases susceptibility. We validated these findings through targeted gene knockouts and gallium sensitivity experiments. Our findings suggest that enterobactin can complex with gallium for cellular uptake, but that the ferric citrate receptor FecA can discriminate between gallium citrate and iron citrate. Expression of fecA increased with gallium exposure, showing that gallium induces FecA-mediated iron uptake. Gallium also increased intracellular levels of manganese in the ΔfecA strain. Supplementation with iron or manganese restored growth of M12 ΔfecA in gallium, suggesting that gallium sensitivity is linked to both iron starvation and oxidative stress. As the ferric dicitrate transport system is an important virulence factor in several extraintestinal infection sites, our results suggest that targeting FecA may increase E. coli susceptibility to gallium while also suppressing virulence.IMPORTANCEEscherichia coli extraintestinal infections that are resistant to traditional antibiotics are associated with more deaths than any other species. Gallium-based therapies may represent a non-antibiotic approach for treating extraintestinal pathogenic E. coli strains that affect both humans and animals. Our results are significant as they show that the enterobactin siderophore and the ferric dicitrate iron transport systems expressed by these bacteria have opposing roles in E. coli gallium sensitivity. These findings could be leveraged to enhance the efficacy of gallium therapeutics.

(Wilson* et al Science 2024 & unpublished work ) Cancer risk is modulated by a complex network of inherited mutations, DNA replication errors, and environmental exposures. Yet, the impact of genetic variation in the immunosurveillance of nascent and established malignancies remains uncertain. Using population-scale data from the UK Biobank and FinnGen, we uncover a striking association between HLA allelic-associated peptidome diversity and reduced lung cancer risk in smokers. Fine-mapping reveals that amino acid heterozygosity in the HLA-II peptide-binding groove significantly contributes to this protective effect. Single-cell analyses further demonstrate that smoking induces proinflammatory lung macrophages and HLA-II+ epithelial cells, emphasizing a dynamic immune-environment interaction. Furthermore, we identify widespread loss of HLA-II heterozygosity (LOH) in lung cancer, favoring alleles with expanded neopeptide repertoires, alongside distinct LOH patterns for HLA-I and HLA-II across cancer types. Building on these insights, we introduce a novel biophysically informed embedding space, developed with state-of-the-art large language models, to represent HLA allelic similarity as a continuous variable. This framework enables precise quantification of risk across diverse cancers. Together, our findings position genetic variation in immunosurveillance as a pivotal determinant of cancer risk, offering new avenues for predictive modeling and therapeutic innovation. US NIH grant R01 CA283469 Alexander and Alexandrine Sinsheimer Foundation Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

IntroductionBovine mastitis caused by Escherichia coli compromises animal health and inflicts substantial product losses in dairy farming. It may manifest as subclinical through severe acute disease and can be transient or persistent in nature. Little is known about bacterial factors that impact clinical outcomes or allow some strains to outcompete others in the mammary gland (MG) environment. Mastitis-associated E. coli (MAEC) may have distinctive characteristics which may contribute to the varied nature of the disease. Given their high levels of intraspecies genetic variability, virulence factors of commonly used MAEC model strains may not be relevant to all members of this group.MethodsIn this study, we sequenced the genomes of 96 MAEC strains isolated from cattle with clinical mastitis (CM). We utilized clinical severity data to perform genome-wide association studies to identify accessory genes associated with strains isolated from mild or severe CM, or with high or low competitive fitness during in vivo competition assays. Genes associated with mastitis pathogens or commensal strains isolated from bovine sources were also identified.ResultsA type-2 secretion system (T2SS) and a chitinase (ChiA) exported by this system were strongly associated with pathogenic isolates compared with commensal strains. Deletion of chiA from MAEC isolates decreased their adherence to cultured bovine mammary epithelial cells.DiscussionThe increased fitness associated with strains possessing this gene may be due to better attachment in the MG. Overall, these results provide a much richer understanding of MAEC and suggest bacterial processes that may underlie the clinical diversity associated with mastitis and their adaptation to this unique environment.

Bovine mastitis caused by Escherichia coli may manifest as subclinical through severe acute disease and can be transient or persistent in nature. Little is known about bacterial factors that impact clinical outcomes or allow some strains to outcompete others in the mammary gland (MG) environment. Mastitis-associated E. coli (MAEC) may have distinctive characteristics which may contribute to the varied nature of the disease. In this study, we sequenced the genomes of 96 MAEC strains isolated from cattle with clinical mastitis (CM). We utilized clinical severity data to perform genome-wide association studies to identify accessory genes associated with strains isolated from mild or severe CM, or with high or low competitive fitness during in vivo competition assays. Genes associated with pathogenic or commensal strains isolated from bovine and avian sources were also identified. A type-2 secretion system (T2SS) and a chitinase (ChiA) exported by this system were strongly associated with pathogenic isolates compared with commensal strains. Strains carrying these genes also had higher competitive fitness during experimental intramammary infections. Deletion of chiA from MAEC isolates decreased their adherence to cultured bovine mammary epithelial cells, suggesting that the increased fitness associated with strains possessing this gene may be due to better attachment in the MG. Importance Bovine mastitis caused by MAEC compromises animal health and inflicts substantial product losses in dairy farming. Given their high levels of intraspecies genetic variability, virulence factors of commonly used MAEC model strains may not be relevant to all members of this group. Here we analyzed clinical data as well as fitness (quantified in a mouse MG model) of diverse MAEC isolates to identify accessory genes that contribute to infection. We demonstrated a novel role for chitinase in promoting attachment to mammary epithelial cells. Reverse genetic approaches can be applied to the collection of strains and their complete genome sequences that we have presented here. Overall, these results provide a much richer understanding of MAEC and suggest bacterial processes that may underlie the clinical diversity associated with mastitis and their adaptation to this unique environment.

Turkeys (Meleagris gallopavo) provide a globally important source of protein and constitute the second most important source of poultry meat in the world. Bacterial diseases are common in commercial poultry production, causing significant production losses for farmers. Due to the increasingly recognized problems associated with large-scale/indiscriminate antibiotic use in agricultural settings, poultry producers need alternative methods to control common bacterial pathogens. In this study, we compared the cecal microbiota of wild and domestic turkeys, hypothesizing that environmental pressures faced by wild birds may select for a disease-resistant microbial community. Sequence analyses of 16S rRNA genes amplified from cecal samples indicate that free-roaming wild turkeys carry a rich and variable microbiota compared to domestic turkeys raised on large-scale poultry farms. Wild turkeys also had very low levels of Staphylococcus, Salmonella, and Escherichia coli compared to domestic turkeys. E. coli strains isolated from wild and domestic turkey cecal samples also belong to distinct phylogenetic backgrounds and differ in their propensity to carry virulence genes. E. coli strains isolated from factory-raised turkeys were far more likely to carry genes for capsule (kpsII and kpsIII) or siderophore (iroN and fyuA) synthesis than were those isolated from wild turkeys. These results suggest that the microbiota of wild turkeys may provide colonization resistance against common poultry pathogens. IMPORTANCE Due to the increasingly recognized problems associated with antibiotic use in agricultural settings, poultry producers need alternative methods to control common bacterial pathogens. In this study, we compare the microbiota of wild and domestic turkeys. The results suggest that free-ranging wild turkeys carry a distinct microbiome compared to farm-raised turkeys. The microbiome of wild birds contains very low levels of poultry pathogens compared to that of farm-raised birds. The microbiomes of wild turkeys may be used to guide the development of new ways to control disease in large-scale poultry production.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are major causes of urinary and bloodstream infections. ExPEC reservoirs are not completely understood. Some mastitis-associated E. coli (MAEC) strains carry genes associated with ExPEC virulence, including metal scavenging, immune avoidance, and host attachment functions. In this study, we investigated the role of the high-affinity zinc uptake (znuABC) system in the MAEC strain M12. Elimination of znuABC moderately decreased fitness during mouse mammary gland infections. The ΔznuABC mutant strain exhibited an unexpected growth delay in the presence of bile salts, which was alleviated by the addition of excess zinc. We isolated suppressor mutants with improved growth in bile salts, several of which no longer produced the K96 capsule made by strain M12. The addition of bile salts also reduced capsule production by strain M12 and ExPEC strain CP9, suggesting that capsule synthesis may be detrimental when bile salts are present. To better understand the role of the capsule, we compared the virulence of mastitis strain M12 with that of its unencapsulated ΔkpsCS mutant in two models of ExPEC disease. The wild-type strain successfully colonized mouse bladders and kidneys and was highly virulent in intraperitoneal infections. Conversely, the ΔkpsCS mutant was unable to colonize kidneys and was unable to cause sepsis. These results demonstrate that some MAEC strains may be capable of causing human ExPEC illness. The virulence of strain M12 in these infections is dependent on its capsule. However, capsule may interfere with zinc homeostasis in the presence of bile salts while in the digestive tract.

ABSTRACT Extraintestinal pathogenic Escherichia coli (ExPEC) are major causes of urinary and bloodstream infections. ExPEC reservoirs are not completely understood. Some mastitis-associated E. coli (MAEC) strains carry genes associated with ExPEC virulence, including metal scavenging, immune avoidance, and host attachment functions. In this study, we investigated the role of the high-affinity zinc uptake ( znuABC ) system in the MAEC strain M12. Elimination of znuABC moderately decreased fitness during mouse mammary gland infections. The Δ znuABC mutant strain exhibited an unexpected growth delay in the presence of bile salts, which was alleviated by the addition of excess zinc. We isolated Δ znuABC mutant suppressor mutants with improved growth of in bile salts, several of which no longer produced the K96 capsule made by strain M12. Addition of bile salts also reduced capsule production by strain M12 and ExPEC strain CP9, suggesting that capsule synthesis may be detrimental when bile salts are present. To better understand the role of the capsule, we compared the virulence of mastitis strain M12 with its unencapsulated Δ kpsCS mutant in two models of ExPEC disease. The wild type strain successfully colonized mouse bladders and kidneys and was highly virulent in intraperitoneal infections. Conversely, the Δ kpsCS mutant was unable to colonize kidneys and was unable to cause sepsis. These results demonstrate that some MAEC may be capable of causing human ExPEC illness. Virulence of strain M12 in these infections is dependent on its capsule. However, capsule may interfere with zinc homeostasis in the presence of bile salts while in the digestive tract.

A recent genome-wide association study (GWAS) of 59 cerebrospinal fluid (CSF) proteins with a connection to Alzheimer's disease (AD) demonstrated an association between increased levels of chemokine ligand 2 (CCL2) with an atypical chemokine receptor chemokine-binding protein 2 variant V41A (ACKR2-V41A; rs2228467). High levels of CCL2 are associated with increased risk of AD development as well as other inflammatory diseases. In this study we characterized the biological function of the ACKR2-V41A receptor compared to the wild type allele by measuring its ligand binding affinity, CCL2 scavenging efficiency, and cell activation sensitivity. We transfected Chinese hamster ovary cells with plasmids carrying wild type ACKR2 (ACKR2-WT) or the mutant ACKR2-V41A receptor. Binding affinity assays showed that ACKR2-V41A has a lower binding affinity for CCL2 and CCL4 than ACKR2-WT. CCL2 scavenging results aligned with binding affinity assays, with ACKR2-V41A cells scavenging CCL2 with a lower efficiency than ACKR2-WT. Cell activation assays also showed that ACKR2-V41A cells had significantly lower receptor upregulation (β-Arrestin-dependent signaling pathway) upon stimulation compared to ACKR2-WT cells. These findings provide molecular and biological mechanistic insights into the GWAS association of ACKR2-V41A with increased levels of CCL2 in CSF and possibly other chemokine ligands. Increased CCL2 levels are associated with accelerated cognitive decline and increased risk of AD. Understanding how this atypical chemokine receptor allele increases serum markers of inflammation could lead to novel therapeutic solutions for AD.

Mastitis, resulting from mammary gland infection, is a common and painful disease associated with lactation. In addition to the impact on human and animal health, mastitis causes substantial economic losses in the dairy industry. Staphylococcus aureus is a frequent cause of mastitis worldwide. Despite significant progress in understanding S. aureus pathogenesis in general, much remains to be learned regarding virulence factors relevant in the context of mastitis. This review outlines the molecular mechanisms by which S. aureus acquires essential metals such as iron, zinc, manganese, copper, cobalt and nickel within lactating mammary glands, while exposing areas where our current knowledge is deficient. Increased understanding of how these factors facilitate bacterial survival in the lactating mammary gland can provide therapeutic targets for more effective mastitis prevention and treatment.

In a recent genome-wide association study (GWAS) of 59 cerebrospinal fluid proteins with a connection to the Alzheimer's disease (AD), nonsynonymous MMP3-A133C and nonsynonymous IL6R-G643A showed significant association with higher levels of protein expression. Higher levels of MMP3 and IL6R has been correlated with an increased risk of developing AD. In this study we are replicating these findings, characterizing avidity, affinity, and cell activation between wildtype and mutant. We will transfect Chinese hamster ovary cells (CHOK1) with plasmids carrying a wildtype or mutant plasmid of IL6R and MMP3. We will test binding affinity of the receptor using their respective ligands. In addition, we will seek to observe differences in the receptor's avidity and scavenging, cell activation. GWAS analysis have indicated that MMP3-A133C and IL6R-G643A are associated with increased risk of developing AD. Results of the molecular characterization will be disclosed during the conference. This work will provide insights into the association of MMP3-A133C and IL6R-G643A with AD risk, rate of cognitive decline, and serum markers of inflammation which could lead to novel therapeutic solutions for AD.

In a recent genome-wide association study (GWAS) of 59 cerebrospinal fluid proteins with a connection to the Alzheimer's disease (AD), the nonsynonymous mutations CCBP2-V41A (rs2228467) showed significant association with higher levels of the C-C chemokine ligand motif 2 (CCL2). It is known that a high level of CCL2 is associated with risk development of AD. In this study we are replicating this finding, characterizing avidity, scavenging, and cell activation between wildtype and mutant. We have transfected Chinese hamster ovary cells (CHOK1) with plasmids carrying a wildtype or mutant version of CCBP2. We have tested the binding ability of the receptor using CCL2. We will test for differences in receptor avidity and scavenging, and cell activation. Receptor CCBP2-wildtype has a higher binding avidity with CCL2 than CCBP2-V41A (p-value 0.045; 9 samples), making these results consistent with GWAS analysis previously done. There is not chemical difference at the SNP site. Results for avidity, scavenging, and cell activation assays as well as association analysis will be reported at the conference.

In a recent genome-wide association study (GWAS) of 59 cerebrospinal fluid proteins with a connection to the Alzheimer's disease (AD), the nonsynonymous mutations CCRL2-V180M (rs6441977) showed significant association with lower levels of the C-C chemokine ligand motif 4 (CCL4). It is known that a high level of CCL4 is associated with risk development of AD, suggesting that this mutation can be a possible resilience marker for AD. In this study we are characterizing the biological basis of this finding characterizing avidity, scavenging, and cytokine association between wildtype and mutant genotypes at rs6441977. We have transfected Chinese hamster ovary cells (CHOK1) with plasmids carrying a wildtype or mutant version of CCRL2. We have tested the binding ability of the receptor using Chemerin, its natural ligand. We will test for differences in receptor avidity and scavenging, cytokine associations. Receptor CCRL2-V180M has a higher binding assay with Chemerin than wildtype (p-value 0.03; 15 samples). There is a chemical change at the SNP site. CCL4 does not interfere with interaction between CCRL2 and Chemerin. Results for avidity and scavenging assays as well as an association analysis will be reported at the conference.