Biology subjects

Mobley, H. L. T.

Publications and source records attributed to Mobley, H. L. T..

13 recordsLinked to original sources

bioRxiv2024

Multiplexed PCR to measure in situ growth rates of uropathogenic E. coli during experimental urinary tract infection.

Measuring bacterial growth rates in vitro is routine, however, determining growth rates during infection within a host has been more challenging. Peak-to-trough ratio (PTR) is a technique for studying microbial growth dynamics, calculated using the ratio of replication origin (ori) copies to those of the terminus (ter), as originally defined by whole genome sequencing (WGS). WGS presents significant challenges in terms of expense and data analysis complexity due to the presence of host DNA in the samples. Here, we used multiplexed PCR with fluorescent probes to estimate bacterial growth rates based on the abundance of ori- and ter-adjacent loci, without the need for WGS. We establish the utility of this approach by comparing growth rates of the uropathogenic Escherichia coli (UPEC) strain HM86 by WGS (PTR) and qPCR to measure the equivalent ori:ter (O:TPCR). We found that PTR and O:TPCR were highly correlated and that O:TPCR reliably predicted growth rates calculated by conventional methods. O:TPCR was then used to calculate the in situ E. coli growth rates in urine, bladder, and kidneys collected over the course of a week from a murine model of urinary tract infection (UTI). These analyses revealed that growth rate of UPEC strains gradually increased during the early stages of infection (0-6h), followed by a slow decrease in growth rates during later time points (1-7 days). This rapid and convenient method provides valuable insights into bacterial growth dynamics during infection and can be applied to other bacterial species in both animal models and clinical infections. ImportanceAccurately measuring bacterial growth rates in the host, which plays a crucial role in determining the success of pathogens in establishing infections, poses significant challenges. To address this, bacterial replication rate has been measured as a proxy for growth rate estimation. While whole genome sequencing (WGS) has been used for this purpose, it comes with drawbacks such as high costs and difficulties in analyzing bacterial sequences due to the overwhelming presence of host DNA. In this study, we validate a more accessible PCR-based approach compared to the established WGS method and confirmed the reliability of our PCR-based technique. We then applied it to measure the growth rate of Escherichia coli during experimental urinary tract infection in a mouse model. This study provides a cost-effective and efficient alternative to WGS for studying bacterial replication dynamics during infection, potentially offering new insights into pathogen behavior and host-microbe interactions.

microbiology↗

bioRxiv2024

Infection characteristics among Serratia marcescens capsule lineages.

Serratia marcescens is a healthcare-associated pathogen that can cause severe infections including bacteremia and pneumonia. The capsule polysaccharide of S. marcescens is a bacteremia fitness determinant and previous work defined capsule locus (KL) diversity within the species. Strains belonging to KL1 and KL2 capsule clades produce sialylated polysaccharides and represent the largest subpopulation of isolates from clinical origin. In this study, the contribution of these and other S. marcescens capsules to infection was determined in animal and cellular models. Using a murine model of primary bacteremia, clinical isolates of multiple KL types demonstrated capsule-dependent colonization of spleen, liver, and kidney following tail vein inoculation. Similar results were observed using a bacteremic pneumonia model, in that all tested strains of clinical origin demonstrated a requirement for capsule in both the primary lung infection site and for bloodstream dissemination to secondary organs. Finally, capsule from each KL clade was examined for the ability to resist internalization by bone marrow-derived macrophages. Only the sialylated KL1 and KL2 clade strains exhibited capsule-dependent inhibition of internalization, including KL2 capsule produced in a heterologous background. Together these findings indicate that lineage-specific resistance to macrophage phagocytosis may enhance survival and antibacterial defenses of clinically-adapted S. marcescens. IMPORTANCEBacteremia occurs when the host immune system fails to contain bacterial bloodstream replication following an initial inoculation event from either an internal or external source. Capsule polysaccharides play a protective role for Serratia marcescens during bacteremia but there is abundant genetic diversity at the capsule-encoding locus within the species. This study compares the infection characteristics of S. marcescens isolates belonging to five capsule types and defines the contributions to infection fitness for each. By characterizing the differences in capsule dependence and infection potential between S. marcescens strains, efforts to combat these life-threatening infections can be focused toward identifying strategies that target the most critical genetic lineages of this important opportunistic pathogen.

microbiology↗

bioRxiv2024

A novel method for integrating genomic and Tn-Seq data to identify common in vivo fitness mechanisms across multiple bacterial species

Sepsis is life-threatening organ dysfunction due to an unregulated immune response to infection. Bacteremia is a leading cause of sepsis, and members of the Enterobacterales cause nearly half of bacteremia cases annually. While previous Tn-Seq studies to identify novel bacteremia-fitness genes have provided valuable insight into virulence mechanisms, evidence for common pathways across species is lacking. To identify common fitness pathways in five bacteremia- caused Enterobacterales species, we utilized the JCVI pan-genome pipeline to integrate Tn-Seq fitness data with multiple available functional data types. Core genes from species pan-genomes were used to construct a multi-species core pan-genome, producing 2,850 core gene clusters found in four out of the five species. Integration of Tn-Seq fitness data enabled identification of 373 protein clusters that were conserved in all five species. A scoring rubric and filter was applied to these clusters, which incorporated Tn-Seq fitness defects, operon localization, and antibiotic susceptibility data, which reduced the number of bacteremia-fitness genes and identified seven common fitness mechanisms. Independent mutational validation of one prioritized fitness gene, tatC, showed reduced fitness in vivo and increased susceptibility to beta- lactams that were restored following tatC complementation in trans. By integrating known operon structures and antibiotic susceptibility with Tn-Seq fitness data, common genes within the core pan-genome emerged and revealed mechanisms that are essential for colonization of, or survival in, the mammalian bloodstream. Our prediction and validation of tatC as a common bacteremia fitness factor and contributor of antibiotic resistance supports the utility of this bioinformatic approach. This study represents a major step forward to prioritize potentially novel targets for therapy against these deadly widespread sepsis infections. Author SummaryBacteremia is a leading cause of sepsis, a life-threatening condition where an unregulated immune response to infection causes systemic organ failure. Nearly half of bacteremia cases are caused by members of the Gram-negative bacterial taxonomic order Enterobacterales. Given the public health impact of bacteremia and the reduction of existing antibiotic treatment options, novel strategies are needed to combat these infections. Pan-genome software was used to predict seven shared fitness pathways in these bacteria that may serve as novel targets for treatment of bacteremia. Briefly, a scoring rubric was applied to shared pan-genome clusters, which incorporated multiple data types, including Tn-Seq fitness defects, operon localization, and antibiotic susceptibility data to rank and prioritize fitness genes. To validate one of our predictions, mutations were constructed in tatC, which showed both reduced fitness in mice and increased susceptibility to beta-lactam antibiotics; complementation restored fitness and antibiotic susceptibility to wild type levels. This study takes a novel bioinformatics approach to build a core pan-genome across multiple distantly related bacteria to integrate computational and experimental data to predict important shared fitness genes and represents a major step forward toward identifying novel targets of therapy against these deadly widespread life-threatening infections.

bioinformatics↗

bioRxiv2024

Fitness Factor Genes Conserved within the Multi-species Core Genome of Gram-negative Enterobacterales Species Contribute to Bacteremia Pathogenesis

There is a critical gap in knowledge about how Gram-negative bacterial pathogens, using survival strategies developed for other niches, cause lethal bacteremia. Facultative anaerobic species of the Enterobacterales order are the most common cause of Gram-negative bacteremia, including Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, and Enterobacter hormaechei. Bacteremia often leads to sepsis, a life-threatening organ dysfunction resulting from an unregulated immune response to infection. Despite a lack of specialization for this host environment, Gram-negative pathogens cause nearly half of bacteremia cases annually. Based on our existing Tn-Seq fitness factor data from a murine model of bacteremia combined with comparative genomics of the five Enterobacterales species above, we prioritized 18 conserved fitness genes or operons for further characterization. Each mutant in each species was used to cochallenge C57BL/6 mice via tail vein injection along with the respective wild-type strain to determine competitive indices for each fitness gene or operon. Among the five species, we found three fitness factor genes, that when mutated, attenuated the mutant for all species in the spleen and liver (tatC, ruvA, gmhB). Nine additional fitness factor genes or operons were validated as outcompeted by wild-type in three or four bacterial species in the spleen (xerC, wzxE, arcA, prc, apaGH, atpG, lpdA, ubiH, aroC). Overall, 17 of 18 fitness factor mutants were attenuated in at least one species in the spleen or liver. Together, these findings allow for the development of a model of bacteremia pathogenesis that may include future targets of therapy against bloodstream infections. >Author SummaryFrequent cases of bacteremia plague our ICUs, bone marrow transplant units, and inpatient facilities. Nearly half of these infections are caused by Gram-negative bacteria. The Enterobacterales order including E. coli, K. pneumoniae, S. marcescens, C. freundii, and E. hormaechei are leading causes of bacteremia. An alarming proportion of these are due to antibiotic-resistant isolates, which are four times more likely to kill than antibiotic-susceptible isolates. Clearly, we need new therapeutic targets to treat cases of bacteremia and sepsis. Previously, it has been unclear what genes contribute to their ability to survive in this hostile host environment. We have previously undertaken unbiased genetic screens to identify 18 genes shared by all five bacterial genera that are required for survival in blood and blood-filtering organs. These include genes that encode proteins that maintain proton motive force, resist antimicrobial peptides and complement, mediate genome maintenance, transport key metabolites and proteins, avoid oxidative stress, acquire iron, and regulate key pathways. Mutants, constructed in these shared genes in the five species, were validated for a high proportion of genes as critical for infection in the mouse model of bacteremia.

microbiology↗

bioRxiv2023

Altered motility in response to iron-limitation is regulated by lpdA in uropathogenic E. coli CFT073

More than half of all women will experience a urinary tract infection (UTI) in their lifetime with most cases caused by uropathogenic Escherichia coli (UPEC). Bacterial motility enhances UPEC pathogenicity, resulting in more severe disease outcomes including kidney infection. Surprisingly, the connection between motility and iron limitation is mostly unexplored, despite the lack of free iron available in the host. Therefore, we sought to explore the potential connection between iron restriction and regulation of motility in UPEC. We cultured E. coli CFT073, a prototypical UPEC strain, in media containing an iron chelator. Under iron limitation, CFT073 had elevated fliC (flagella) promoter activity, driving motility on the leading edge of the colony. Furthermore, this iron-specific response was repressed by the addition of exogenous iron. We confirmed increased flagella expression in CFT073 by measuring fliC transcript, FliC protein, and surface-expressed flagella under iron-limited conditions. To define the regulatory mechanism, we constructed single knockouts of eight master regulators. The iron-regulated response was lost in crp, arcA, and fis mutants. Thus, we focused on the five genes regulated by all three transcription factors. Of the five genes knocked out, the iron-regulated motility response was most strongly dysregulated in an lpdA mutant, which also resulted in significantly lowered fitness in the murine model of ascending UTI. Collectively, we demonstrated that iron-mediated motility in CFT073 is regulated by lpdA, which contributes to the understanding of how uropathogens differentially regulate motility mechanisms in the iron-restricted host. ImportanceUrinary tract infections (UTIs) are ubiquitous and responsible for over five billion dollars in associated health care costs annually. Both iron acquisition and motility are highly studied virulence factors associated with uropathogenic E. coli (UPEC), the main causative agent of uncomplicated UTI. This work is innovative by providing mechanistic insight into the synergistic relationship between these two critical virulence properties. Here, we demonstrate that iron limitation has pleiotropic effects with consequences that extend beyond metabolism, and impact other virulence mechanisms. Indeed, targeting iron acquisition as a therapy may lead to an undesirable enhancement of UPEC pathogenesis through increased motility. It is vital to understand the full breadth of UPEC pathogenesis to adequately respond to this common infection, especially with the increase of antibiotic resistant pathogens.

microbiology↗

bioRxiv2023

Conserved metabolic regulator ArcA responds to oxygen availability, iron limitation, and cell envelope perturbations during bacteremia

Bacteremia, a systemic infection associated with severe clinical outcomes, is often caused by Gram-negative facultative anaerobes. ArcAB, a two-component regulatory system that represses aerobic respiration, is a key mediator of metabolic adaptation for such bacteria. Using targeted mutational analysis informed by global genetic screens, we identified the arcA gene as promoting fitness of Klebsiella pneumoniae, Citrobacter freundii, and Serratia marcescens but not Escherichia coli in a murine model of bacteremia. Engineered mutants lacking arcA exhibit a dysregulated response to changes in oxygen availability, iron limitation, and membrane perturbations, all of which bacterial cells experience during infection. The genetic response of the arcA mutants relative to wild-type strains to the cationic antimicrobial peptide polymyxin B demonstrates an expanded role for ArcA as an activator in response to membrane damage in addition to metabolic adaptation. ArcA function is furthermore linked to electron transport chain activity based on its response to uncoupling of proton motive force by carbonyl cyanide-m-chlorophenylhydrazone (CCCP). Differences in lactate and acetate levels as well as lactate dehydrogenase activity between arcA mutant and wild-type cells following CCCP treatment establish an ArcA-mediated shift to fermentation independent of oxygen availability. This study highlights the semi-conserved role of ArcA during bacteremia and consolidates infection phenotypes into a comprehensive model based on respiratory activity. AUTHOR SUMMARYInfections of the bloodstream are life-threatening and can result in sepsis, an overreaction of the host immune system that ultimately damages the body. Gram-negative bacteria are responsible for causing many cases of bloodstream infections, also referred to as bacteremia. The long-term goal of our work is to understand how these bacteria establish and maintain infection during bacteremia. We have previously identified the transcription factor ArcA, which promotes fermentation in bacteria, as a likely contributor to the growth and survival of bacteria in this environment. Here, we study ArcA in the Gram-negative species Citrobacter freundii, Klebsiella pneumoniae, and Serratia marcescens. Our findings aid in determining how these bacteria sense their environment, utilize nutrients, and generate energy while also countering attacks from the host immune system. This information is critical for developing better models of infection to inform future therapeutic development.

microbiology↗

bioRxiv2023

Klebsiella pneumoniae causes bacteremia using factors that mediate tissue-specific fitness and resistance to oxidative stress

Gram-negative bacteremia is a major cause of global morbidity involving three phases of pathogenesis: initial site infection, dissemination, and survival in the blood and filtering organs. Klebsiella pneumoniae is a leading cause of bacteremia and pneumonia is often the initial infection. In the lung, K. pneumoniae relies on many factors like capsular polysaccharide and branched chain amino acid biosynthesis for virulence and fitness. However, mechanisms directly enabling bloodstream fitness are unclear. Here, we performed transposon insertion sequencing (TnSeq) in a tail-vein injection model of bacteremia and identified 58 K. pneumoniae bloodstream fitness genes. These factors are diverse and represent a variety of cellular processes. In vivo validation revealed tissue-specific mechanisms by which distinct factors support bacteremia. ArnD, involved in Lipid A modification, was required across blood filtering organs and supported resistance to soluble splenic factors. The purine biosynthesis enzyme PurD largely enhanced liver fitness and was required for replication in serum. PdxA, a member of the endogenous vitamin B6 biosynthesis pathway, optimized replication in serum and lung fitness. The stringent response regulator SspA was required for splenic fitness yet was dispensable in the liver. In a bacteremic pneumonia model that incorporates initial site infection and dissemination, splenic fitness defects were enhanced, and DsbA, SspA, and PdxA increased fitness across bacteremia phases. SspA and PdxA enhanced K. pnuemoniae resistance to oxidative stress. SspA specifically resists oxidative stress produced by NADPH oxidase Nox2 in the lung, spleen, and liver, as it was a fitness factor in wild-type but not Nox2-deficient (Cybb-/-) mice. These results identify site-specific fitness factors that act during the progression of Gram-negative bacteremia. Defining K. pneumoniae fitness strategies across bacteremia phases could illuminate therapeutic targets that prevent infection and sepsis. Author SummaryGram-negative bacteremia is a deadly family of infections that initiate sepsis, a leading cause of global morbidity and mortality. Only a small number of Gram-negative species contribute to the majority of clinical bacteremia. Klebsiella pneumoniae is the second leading cause of Gram-negative bacteremia, and the third leading cause of overall bloodstream infection. K. pneumoniae is highly linked to hospital-associated infection with increasing antimicrobial resistance, endangering the most vulnerable patients. It is critical to understand the pathogenesis of K. pneumoniae bacteremia to better develop targets for future therapies that can prevent these deadly infections. Here, we define over 50 K. pneumoniae genes that support bloodstream fitness. These factors are diverse, support tissue-specific fitness, and increase bacterial resistance to oxidative stress. Our study is the first to systematically define K. pneumoniae factors enhancing bacteremia in a mammalian system. These results illuminate host-pathogen interactions during K. pneumoniae bacteremia that may be extended to additional Gram-negative species.

microbiology↗

bioRxiv2022

Phenotypic Assessment of Clinical Escherichia coli Isolates Predicts Uropathogenic Potential

For women in the United States, urinary tract infections (UTI) are the most frequent diagnosis in emergency departments, comprising 21.3% of total visits. Uropathogenic Escherichia coli (UPEC) causes [~]80% of uncomplicated UTI. To combat this public health issue, it is vital to characterize UPEC strains as well as differentiate them from commensal strains to reduce the overuse of antibiotics. Surprisingly, no genetic signature has been identified which clearly separates UPEC from other E. coli. Therefore, we examined whether phenotypic data could be predictive of uropathogenic potential. We screened 13 clinical strains of UPEC, isolated from cases of uncomplicated UTI in young otherwise healthy women, in a series of microbiological phenotypic assays using UPEC prototype strain CFT073 and non-pathogenic E. coli strain MG1655 K12 as controls. Phenotypes included adherence, iron acquisition, biofilm formation, human serum resistance, motility, and stress resistance. These data were able to predict the severity of bacterial burden in both the urine and bladders using a well-established experimental mouse model of UTI. Multiple linear regression using three different phenotypic assays, growth in minimal medium, siderophore production, and type 1 fimbrial expression, was predictive of bladder colonization (adjusted r2=0.6411). Growth in ex vivo human urine, hemagglutination of red blood cells, and motility modeled urine colonization (adjusted r2=0.4821). These results showcase the utility of phenotypic characterization to predict the severity of infection these strains may cause. We predict that these methods will also be applicable to other complex, genetically redundant, pathogens. ImportanceUrinary tract infections are the second leading infectious disease worldwide, occurring in over half of the female population during their lifetime. Most infections are caused by uropathogenic Escherichia coli (UPEC). These strains can commensally colonize the gut, but upon introduction to the urinary tract, can infect the host and cause disease. Clinically, it would be beneficial to screen patient E. coli strains to understand their pathogenic potential, which may lead to the administration of prophylactic antibiotic treatment for those with increased risk. Others have proposed the use of PCR-based genetic screening methods to detect UPEC and differentiate them from other E. coli pathotypes; however, this method has not yielded a consistent uropathogenic signature. Here, we have used phenotypic characteristics such as growth rate, siderophore production, and expression of fimbriae to successfully predict uropathogenic potential.

microbiology↗

bioRxiv2021

Ferric Citrate Uptake is a Virulence Factor in Uropathogenic Escherichia coli

More than half of women will experience a urinary tract infection (UTI) with uropathogenic Escherichia coli (UPEC) causing [~]80% of uncomplicated cases. Iron acquisition systems are essential for uropathogenesis, and UPEC encode functionally redundant iron acquisition systems, underlining their importance. However, a recent UPEC clinical isolate, HM7 lacks this functional redundancy and instead encodes a sole siderophore, enterobactin. To determine if E. coli HM7 possesses unidentified iron acquisition systems, we performed RNA-sequencing under iron-limiting conditions and demonstrated that the ferric citrate uptake system (fecABCDE and fecIR) was highly upregulated. Importantly, there are high levels of citrate within urine, some of which is bound to iron, and the fec system is highly enriched in UPEC isolates compared to environmental or fecal strains. Therefore, we hypothesized that HM7 and other similar strains use the fec system to acquire iron in the host. Deletion of both enterobactin biosynthesis and ferric citrate uptake ({Delta}entB/{Delta}fecA) abrogates use of ferric citrate as an iron source and fecA provides an advantage in human urine in absence of enterobactin. However, in a UTI mouse model, fecA is a fitness factor independent of enterobactin production, likely due to the action of host Lipocalin-2 chelating ferrienterobactin. These findings indicate that ferric citrate uptake is used as an iron source when siderophore efficacy is limited, such as in the host during UTI. Defining these novel compensatory mechanisms and understanding the nutritional hierarchy of preferred iron sources within the urinary tract are important in the search for new approaches to combat UTI. ImportanceUPEC, the primary causative agent of uncomplicated UTI is responsible for five billion dollars in healthcare costs in the US each year. Rates of antibiotic resistance are on the rise therefore it is vital to understand the mechanisms of UPEC pathogenesis to uncover potential targets for novel therapeutics. Iron acquisition systems used to obtain iron from sequestered host sources are essential for UPEC survival during UTI and have been used as vaccine targets to prevent infection. This study reveals the ferric citrate uptake system is another important iron acquisition system that is highly enriched in UPEC strains. Ferric citrate uptake has not previously been associated with these pathogenic isolates, underlining the importance of the continued study of these strains to fully understand their mechanisms of pathogenesis.

microbiology↗

bioRxiv2020

A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impact Klebsiella pneumoniae fitness

Hypervirulent K. pneumoniae (hvKp) is a distinct pathotype that causes invasive community-acquired infections in healthy individuals. Hypermucoviscosity (hmv) is a major phenotype associated with hvKp characterized by copious capsule production and poor sedimentation. Dissecting the individual functions of CPS production and hmv in hvKp has been stymied by the conflation of these two properties. Although hmv requires capsular polysaccharide (CPS) biosynthesis, other cellular factors may also be required and some fitness phenotypes ascribed to CPS may be distinctly attributed to hmv. To address this challenge, we systematically identified genes that impact capsule and hmv. We generated a condensed, ordered transposon library in hypervirulent strain KPPR1, then evaluated the CPS production and hmv phenotypes of the 3,733 transposon mutants, representing 72% of all open reading frames in the genome. We employed forward and reverse genetic screens to evaluate effects of novel and known genes on CPS biosynthesis and hmv. These screens expand our understanding of core genes that coordinate CPS biosynthesis and hmv, as well as identify central metabolism genes that distinctly impact CPS biosynthesis or hmv, specifically those related to purine metabolism, pyruvate metabolism and the TCA cycle. Six representative mutants, with varying levels of CPS production and hmv, were all significantly out-competed by wildtype in a murine model of disseminating pneumonia. This suggests that an optimal balance between cellular energetics, CPS biosynthesis and hmv are required for maximal fitness. Altogether, these data demonstrate that hmv requires both CPS biosynthesis and other cellular factors, and that these processes are integrated into the metabolic status of the cell. Therefore, hvKp may require certain nutrients to fully elaborate its virulence-associated properties to specifically cause deep tissue infections. Author summaryKlebsiella pneumoniae is a common multi-drug resistant hospital-associated pathogen, however some isolates are capable of causing community-acquired infections in otherwise healthy individuals. The strains causing community-acquired infections have some distinguishing characteristics, which include overproduction of capsule and hypermucoviscosity. Hypermucoviscous strains are very tacky and sediment poorly when centrifuged. Historically, hypermucoviscosity has been attributed to overproduction of capsular polysaccharide, but recent data suggest that other factors contribute to this bacterial phenotype. Moreover, it seems that capsule and hypermucoviscosity may have distinct roles in pathogenesis. In this study, we sought to systematically investigate the genes that contribute to capsule and hypermucoviscosity. We found that in most cases, genes coordinately impact both capsule biosynthesis and hypermucoviscosity. Some metabolic genes linked to the TCA cycle, however, only affect one of these properties. Here, we identify that capsule biosynthesis and hypermucoviscosity are tightly tied to central metabolism and that an optimal balance between metabolism, capsule, and hypermucoviscosity are important for in vivo fitness of K. pneumoniae. These results identify genes that can be further probed to dissect how capsule and hypermucoviscosity are coordinated in response to niche-specific nutrients. Such studies will expand our understanding of the factors that drive the pathobiology of hypervirulent K. pneumoniae.

microbiology↗

bioRxiv2020

Optimization of an Experimental Vaccine to Prevent Escherichia coli Urinary Tract Infection

Urinary tract infections (UTI) affect half of all women at least once during their lifetime. The rise in extended-spectrum beta-lactamase-producing strains and potential for carbapenem resistance within uropathogenic Escherichia coli (UPEC), the most common causative agent of UTIs, creates an urgent need for vaccine development. Intranasal immunization of mice with UPEC outer membrane iron receptors, FyuA, Hma, IreA, or IutA, conjugated to cholera toxin, provides protection in the bladder or kidneys when challenged with UPEC CFT073 or 536. Based on these data, we sought to optimize the vaccination route (intramuscular, intranasal, or subcutaneous) in combination with adjuvants suitable for human use including alum, monophosphoryl lipid A (MPLA), unmethylated CpG synthetic oligodeoxynucleotides (CpG), polyinosinic:polycytodylic acid (polyIC), and mutated heat-labile E. coli enterotoxin (dmLT). Mice intranasally vaccinated with dmLT-IutA or dmLT-Hma displayed a significant reduction in bladder colonization (86-fold and 32-fold, respectively) with 40-42% of mice having no detectable colony forming units (CFU). Intranasal vaccination of mice with CpG-IutA and polyIC-IutA significantly reduced kidney colonization (131-fold) and urine CFU (22-fold), respectively. dmLT generated the most consistently robust antibody response in intranasally immunized mice, while MPLA and alum produced greater concentrations of antigen-specific serum IgG with intramuscular immunization. Based on these results, we conclude that intranasal administration of Hma or IutA formulated with dmLT adjuvant provides the greatest protection from UPEC UTI. This study advances our progress toward a vaccine against uncomplicated UTI, which will significantly improve the quality of life for women burdened by recurrent UTI and enable better antibiotic stewardship. ImportanceUrinary tract infections (UTI) are among the most common bacterial infection in humans, affecting half of all women at least once during their lifetimes. The rise in antibiotic resistance and health care costs emphasizes the need to develop a vaccine against the most common UTI pathogen, Escherichia coli. Vaccinating mice intranasally with a detoxified heat-labile enterotoxin and two surface exposed receptors, Hma or IutA, significantly reduced bacterial burden in the bladder. This work highlights progress in the development of a UTI vaccine formulated with adjuvants suitable for human use and antigens that encode outer membrane iron receptors required for infection in the iron-limited urinary tract.

microbiology↗

bioRxiv2020

The Gene Expression Profile of Uropathogenic Escherichia coli in Women with Uncomplicated Urinary Tract Infections Is Recapitulated in the Mouse Model

Uropathogenic Escherichia coli (UPEC) is the primary causative agent of uncomplicated urinary tract infections (UTIs). UPEC fitness and virulence determinants have been evaluated in a variety of laboratory settings that include a well-established mouse model of UTI. However, the extent to which bacterial physiology differs between experimental models and human infections remains largely understudied. To address this important question, we compared the transcriptomes of three different UPEC isolates in human infection and a variety of laboratory conditions including LB culture, filter-sterilized urine culture, and the UTI mouse model. We observed high correlation in gene expression between the mouse model and human infection in all three strains examined (Pearson correlation coefficient of 0.86-0.87). Only 175 of 3,266 (5.4%) genes shared by all three strains had significantly different expression levels, with the majority of them (145 genes) down-regulated in patients. Importantly, gene expression of both canonical virulence factors and metabolic machinery were highly similar between the mouse model and human infection, while the in vitro conditions displayed more substantial differences. Interestingly, comparison of gene expression between the mouse model and human infection hint at differences in bladder oxygenation as well as nutrient composition. In summary, our work strongly validates the continued use of this mouse model for the study of the pathogenesis of human UTI. ImportanceDifferent experimental models have been used to study UPEC pathogenesis including in vitro cultures in different media, tissue culture, as well as mouse models of infection. The latter is especially important since it allows evaluation of mechanisms of pathogenesis and potential therapeutic strategies against UPEC. Bacterial physiology is greatly shaped by environment and it is therefore critical to understand how closely bacterial physiology in any experimental model relates to human infection. In this study, we found a very strong correlation in bacterial gene expression between the mouse model and human UTI using identical strains, suggesting that the mouse model accurately mimics human infection, definitively supporting its continued use in UTI research.

microbiology↗

bioRxiv2019

The Klebsiella pneumoniae citrate synthase gene, gltA, influences site specific fitness during infection

Klebsiella pneumoniae (Kp), one of the most common causes of healthcare-associated infections, increases patient morbidity, mortality and hospitalization costs. Kp must acquire nutrients from the host for successful infection. However, the host is able to prevent bacterial nutrient acquisition through multiple systems, including the innate immune protein lipocalin 2 (Lcn2) that prevents Kp iron acquisition by sequestering the siderophore enterobactin. To identify novel Kp factors that mediate evasion of nutritional immunity during lung infection, we undertook an InSeq study using a pool of >20,000 transposon mutants administered to Lcn2+/+ and Lcn2-/- mice. Comparing mutant frequencies between mouse genotypes, this genome-wide screen identified the Kp citrate synthase GltA as potentially interacting with Lcn2, and this novel finding was independently validated. Interestingly, in vitro studies suggest that this interaction is not direct. Given that GltA is involved in oxidative metabolism, we screened the ability of this mutant to use a variety of carbon and nitrogen sources. The results indicated that the gltA mutant has a distinct amino acid auxotrophy and is unable to use a variety of carbon sources. Specifically, we show that gltA is necessary for growth in bronchioloalveolar lavage fluid, which is amino acid-limited, but dispensable in serum, which is amino acid rich. Deletion of Lcn2 from the host leads to increased amino acid levels in bronchioloalveolar lavage fluid, and thus abrogates the loss of gltA during pneumonia in the Lcn2-/- background. GltA was also required for gut colonization, but dispensable in the bloodstream in a bacteremia model, demonstrating that deletion of gltA leads to an organ-specific fitness defect. Together, this study is the first to mechanistically describe a role for gltA in Kp infection and provide unique insight into how metabolic flexibility impacts bacterial fitness during infection.\n\nAuthor SummaryThe bacteria Klebsiella pneumoniae (Kp) is an important cause of infection in healthcare settings. These infections can be difficult to treat, as they frequently occur in chronically ill patients and the bacteria has the ability to acquire multiple antibiotic resistance markers. Kp is a common colonizer of the intestinal tract in hospitalized patients, and can progress to infections of the bloodstream, respiratory and urinary tract. However, the bacterial factors that allow Kp to replicate in these different body sites is unclear. In this study, we found that the Kp citrate synthase, GltA, enables bacterial replication in the lung and intestine by enhancing the ability of Kp to use diverse nutrients, in a mechanism known as metabolic flexibility. Kp lacking GltA require specific amino acids that are abundant in blood, but not other body sites. The work in this study provides novel insight into why Kp is a successful hospital pathogen that can colonize and infect multiple body sites.

microbiology↗