Emerging roles for ABC transporters as virulence factors in uropathogenic Escherichia coli.

Urinary tract infections (UTI) account for a substantial financial burden globally. Over 75% of UTIs are caused by uropathogenic Escherichia coli (UPEC), which have demonstrated an extraordinarily rapid growth rate in vivo. This rapid growth rate appears paradoxical given that urine and the human urinary tract are relatively nutrient-restricted. Thus, we lack a fundamental understanding of how uropathogens propel growth in the host to fuel pathogenesis. Here, we used large in silico, in vivo, and in vitro screens to better understand the role of UPEC transport mechanisms and their contributions to uropathogenesis. In silico analysis of annotated transport systems indicated that the ATP-binding cassette (ABC) family of transporters was most conserved among uropathogenic bacterial species, suggesting their importance. Consistent with in silico predictions, we determined that the ABC family contributed significantly to fitness and virulence in the urinary tract: these were overrepresented as fitness factors in vivo (37.2%), liquid media (52.3%), and organ agar (66.2%). We characterized 12 transport systems that were most frequently defective in screening experiments by generating in-frame deletions. These mutant constructs were tested in urovirulence phenotypic assays and produced differences in motility and growth rate. However, deletion of multiple transport systems was required to achieve substantial fitness defects in the cochallenge murine model. This is likely due to genetic compensation among transport systems, highlighting the centrality of ABC transporters in these organisms. Therefore, these nutrient uptake systems play a concerted, critical role in pathogenesis and are broadly applicable candidate targets for therapeutic intervention.

Proteus mirabilis UreR coordinates cellular functions required for urease activity.

A hallmark of Proteus mirabilis infection of the urinary tract is the formation of stones. The ability to induce urinary stone formation requires urease, a nickel metalloenzyme that hydrolyzes urea. This reaction produces ammonia as a byproduct, which can serve as a nitrogen source and weak base that raises the local pH. The resulting alkalinity induces the precipitation of ions to form stones. Transcriptional regulator UreR activates expression of urease genes in a urea-dependent manner. Thus, urease genes are highly expressed in the urinary tract where urea is abundant. Production of mature urease also requires the import of nickel into the cytoplasm and its incorporation into the urease apoenzyme. Urease accessory proteins primarily acquire nickel from one of two nickel transporters and facilitate incorporation of nickel to form mature urease. In this study, we performed a comprehensive RNA-seq to define the P. mirabilis urea-induced transcriptome as well as the UreR regulon. We identified UreR as the first defined regulator of nickel transport in P. mirabilis. We also offer evidence for the direct regulation of the Ynt nickel transporter by UreR. Using bioinformatics, we identified UreR-regulated urease loci in 15 Morganellaceae family species across three genera. Additionally, we located two mobilized UreR-regulated urease loci that also encode the ynt transporter, implying that UreR regulation of nickel transport is a conserved regulatory relationship. Our study demonstrates that UreR specifically regulates genes required to produce mature urease, an essential virulence factor for P. mirabilis uropathogenesis. IMPORTANCE: Catheter-associated urinary tract infections (CAUTIs) account for over 40% of acute nosocomial infections in the USA and generate $340 million in healthcare costs annually. A major causative agent of CAUTIs is Proteus mirabilis, an understudied Gram-negative pathogen noted for its ability to form urinary stones via the activity of urease. Urease mutants cannot induce stones and are attenuated in a murine UTI model, indicating this enzyme is essential to P. mirabilis pathogenesis. Transcriptional regulation of urease genes by UreR is well established; here, we expand the UreR regulon to include regulation of nickel import, a function required to produce mature urease. Furthermore, we reflect on the role of urea catalysis in P. mirabilis metabolism and provide evidence for its importance.

Organ agar serves as physiologically relevant alternative for in vivo bacterial colonization.

Animal models for host-microbial interactions have proven valuable, yielding physiologically relevant data that may be otherwise difficult to obtain. Unfortunately, such models are lacking or nonexistent for many microbes. Here, we introduce organ agar, a straightforward method to enable the screening of large mutant libraries while avoiding physiological bottlenecks. We demonstrate that growth defects on organ agar were translatable to bacterial colonization deficiencies in a murine model. Specifically, we present a urinary tract infection agar model to interrogate an ordered library of Proteus mirabilis transposon mutants, with accurate prediction of bacterial genes critical for host colonization. Thus, we demonstrate the ability of ex vivo organ agar to reproduce in vivo deficiencies. Organ agar was also useful for identifying previously unknown links between biosynthetic genes and swarming motility. This work provides a readily adoptable technique that is economical and uses substantially fewer animals. We anticipate this method will be useful for a wide variety of microorganisms, both pathogenic and commensal, in a diverse range of model host species.

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

IMPORTANCE: Infections of the bloodstream are life-threatening and can result in sepsis. Gram-negative bacteria cause a significant portion of bloodstream infections, which is also referred to as bacteremia. The long-term goal of our work is to understand how such 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 countering the host immune system. This information is critical for developing better models of infection to inform future therapeutic development.

Urine-mediated suppression of Klebsiella pneumoniae mucoidy is counteracted by spontaneous Wzc variants altering capsule chain length.

Klebsiella pneumoniae is a hospital-associated pathogen primarily causing urinary tract infections (UTIs), pneumonia, and septicemia. Two challenging lineages include the hypervirulent strains, causing invasive community-acquired infections, and the carbapenem-resistant classical strains, most frequently isolated from UTIs. While hypervirulent strains are often characterized by a hypermucoid phenotype, classical strains usually present with low mucoidy. Since clinical UTI isolates tend to exhibit limited mucoidy, we hypothesized that environmental conditions may drive K. pneumoniae adaptation to the urinary tract and select against mucoid isolates. We found that both hypervirulent K. pneumoniae and classical Klebsiella UTI isolates significantly suppressed mucoidy when cultured in urine without reducing capsule abundance. A genetic screen identified secondary mutations in the wzc tyrosine kinase that overcome urine-suppressed mucoidy. Over-expressing Wzc variants in trans was sufficient to boost mucoidy in both hypervirulent and classical Klebsiella UTI isolates. Wzc is a bacterial tyrosine kinase that regulates capsule polymerization and extrusion. Although some Wzc variants reduced Wzc phospho-status, urine did not alter Wzc phospho-status. Urine does, however, increase K. pneumoniae capsule chain length diversity and enhance cell-surface attachment. The identified Wzc variants counteract urine-mediated effects on capsule chain length and cell attachment. Combined, these data indicate that capsule chain length correlates with K. pneumoniae mucoidy and that this extracellular feature can be fine-tuned by spontaneous Wzc mutations, which alter host interactions. Spontaneous Wzc mutation represents a global mechanism that could fine-tune K. pneumoniae niche-specific fitness in both classical and hypervirulent isolates. IMPORTANCE Klebsiella pneumoniae is high-priority pathogen causing both hospital-associated infections, such as urinary tract infections, and community-acquired infections. Clinical isolates from community-acquired infection are often characterized by a tacky, hypermucoid phenotype, while urinary tract isolates are usually not mucoid. Historically, mucoidy was attributed to capsule overproduction; however, recent reports have demonstrated that K. pneumoniae capsule abundance and mucoidy are not always correlated. Here, we report that human urine suppresses K. pneumoniae mucoidy, diversifies capsule polysaccharide chain length, and increases cell surface association. Moreover, specific mutations in the capsule biosynthesis gene, wzc, are sufficient to overcome urine-mediated suppression of mucoidy. These Wzc variants cause constitutive production of more uniform capsular polysaccharide chains and increased release of capsule from the cell surface, even in urine. These data demonstrate that K. pneumoniae regulates capsule chain length and cell surface attachment in response host cues, which can alter bacteria-host interactions.

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 supported liver fitness in vivo 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. ArnD, PurD, DsbA, SspA, and PdxA increased fitness across bacteremia phases and each demonstrated unique fitness dynamics within compartments in this model. SspA and PdxA enhanced K. pnuemoniae resistance to oxidative stress. SspA, but not PdxA, 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.

Organ agar serves as physiologically relevant alternative for in vivo colonization.

Animal models for host-microbial interactions have proven valuable, yielding physiologically relevant data that may be otherwise difficult to obtain. Unfortunately, such models are lacking or nonexistent for many microbes. Here, we introduce organ agar, a straightforward method to enable the screening of large mutant libraries while avoiding physiological bottlenecks. We demonstrate that growth defects on organ agar were translatable to colonization deficiencies in a murine model. Specifically, we present a urinary tract infection agar model to interrogate an ordered library of Proteus mirabilis transposon mutants, with accurate prediction of bacterial genes critical for host colonization. Thus, we demonstrate the ability of ex vivo organ agar to reproduce in vivo deficiencies. This work provides a readily adoptable technique that is economical and uses substantially fewer animals. We anticipate this method will be useful for a wide variety of microorganisms, both pathogenic and commensal, in a diverse range of model host species.

The Klebsiella pneumoniae ter Operon Enhances Stress Tolerance.

Healthcare-acquired infections are a leading cause of disease in patients that are hospitalized or in long-term-care facilities. Klebsiella pneumoniae (Kp) is a leading cause of bacteremia, pneumonia, and urinary tract infections in these settings. Previous studies have established that the ter operon, a genetic locus that confers tellurite oxide (K2TeO3) resistance, is associated with infection in colonized patients. Rather than enhancing fitness during infection, the ter operon increases Kp fitness during gut colonization; however, the biologically relevant function of this operon is unknown. First, using a murine model of urinary tract infection, we demonstrate a novel role for the ter operon protein TerC as a bladder fitness factor. To further characterize TerC, we explored a variety of functions, including resistance to metal-induced stress, resistance to radical oxygen species-induced stress, and growth on specific sugars, all of which were independent of TerC. Then, using well-defined experimental guidelines, we determined that TerC is necessary for tolerance to ofloxacin, polymyxin B, and cetylpyridinium chloride. We used an ordered transposon library constructed in a Kp strain lacking the ter operon to identify the genes that are required to resist K2TeO3-induced and polymyxin B-induced stress, which suggested that K2TeO3-induced stress is experienced at the bacterial cell envelope. Finally, we confirmed that K2TeO3 disrupts the Kp cell envelope, though these effects are independent of ter. Collectively, the results from these studies indicate a novel role for the ter operon as a stress tolerance factor, thereby explaining its role in enhancing fitness in the gut and bladder.

Construction of an Ordered Transposon Library for Uropathogenic Proteus mirabilis HI4320.

Ordered transposon libraries are a valuable resource for many bacterial species, especially those with difficult methods for generating targeted genetic mutations. Here, we present the construction of an ordered transposon library for the bacterial urinary tract pathogen Proteus mirabilis strain HI4320. This library will facilitate future studies into P. mirabilis biology. For large experimental screens, it may be used to overcome bottleneck constraints and avoid biased outcomes resulting from gene length. For smaller studies, the library allows sidestepping the laborious construction of single targeted mutants. This library, containing 18,432 wells, was condensed into a smaller library containing 1,728 mutants. Each selected mutant had a single transposon insertion in an open reading frame, covering 45% of predicted genes encoded by P. mirabilis HI4320. This coverage was lower than expected and was due both to library wells with no mapped insertions and a surprisingly high proportion of mixed clones and multiple transposon insertion events. We offer recommendations for improving future library construction and suggestions for how to use this P. mirabilis library resource. IMPORTANCE Ordered libraries facilitate large genetic screens by guaranteeing high genomic coverage with a minimal number of mutants, and they can save time and effort by reducing the need to construct targeted mutations. This resource is now available for P. mirabilis, a common and complicating agent of catheter-associated urinary tract infection. We also present obstacles encountered during library construction with the goal to aid others who would like to construct ordered transposon libraries in other species.

The ADP-Heptose Biosynthesis Enzyme GmhB is a Conserved Gram-Negative Bacteremia Fitness Factor.

Klebsiella pneumoniae is a leading cause of Gram-negative bacteremia, which is a major source of morbidity and mortality worldwide. Gram-negative bacteremia requires three major steps: primary site infection, dissemination to the blood, and bloodstream survival. Because K. pneumoniae is a leading cause of health care-associated pneumonia, the lung is a common primary infection site leading to secondary bacteremia. K. pneumoniae factors essential for lung fitness have been characterized, but those required for subsequent bloodstream infection are unclear. To identify K. pneumoniae genes associated with dissemination and bloodstream survival, we combined previously and newly analyzed insertion site sequencing (InSeq) data from a murine model of bacteremic pneumonia. This analysis revealed the gene gmhB as important for either dissemination from the lung or bloodstream survival. In Escherichia coli, GmhB is a partially redundant enzyme in the synthesis of ADP-heptose for the lipopolysaccharide (LPS) core. To characterize its function in K. pneumoniae, an isogenic knockout strain (ΔgmhB) and complemented mutant were generated. During pneumonia, GmhB did not contribute to lung fitness and did not alter normal immune responses. However, GmhB enhanced bloodstream survival in a manner independent of serum susceptibility, specifically conveying resistance to spleen-mediated killing. In a tail-vein injection of murine bacteremia, GmhB was also required by K. pneumoniae, E. coli, and Citrobacter freundii for optimal fitness in the spleen and liver. Together, this study identifies GmhB as a conserved Gram-negative bacteremia fitness factor that acts through LPS-mediated mechanisms to enhance fitness in blood-filtering organs.

Augmented Enterocyte Damage During Candida albicans and Proteus mirabilis Coinfection.

The human gut acts as the main reservoir of microbes and a relevant source of life-threatening infections, especially in immunocompromised patients. There, the opportunistic fungal pathogen Candida albicans adapts to the host environment and additionally interacts with residing bacteria. We investigated fungal-bacterial interactions by coinfecting enterocytes with the yeast Candida albicans and the Gram-negative bacterium Proteus mirabilis resulting in enhanced host cell damage. This synergistic effect was conserved across different P. mirabilis isolates and occurred also with non-albicans Candida species and C. albicans mutants defective in filamentation or candidalysin production. Using bacterial deletion mutants, we identified the P. mirabilis hemolysin HpmA to be the key effector for host cell destruction. Spatially separated coinfections demonstrated that synergism between Candida and Proteus is induced by contact, but also by soluble factors. Specifically, we identified Candida-mediated glucose consumption and farnesol production as potential triggers for Proteus virulence. In summary, our study demonstrates that coinfection of enterocytes with C. albicans and P. mirabilis can result in increased host cell damage which is mediated by bacterial virulence factors as a result of fungal niche modification via nutrient consumption and production of soluble factors. This supports the notion that certain fungal-bacterial combinations have the potential to result in enhanced virulence in niches such as the gut and might therefore promote translocation and dissemination.

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 strains encode highly diverse iron acquisition systems, underlining their importance. However, a recent UPEC clinical isolate, HM7, lacks this diversity and instead encodes the synthesis pathway for a sole siderophore, enterobactin. To determine if 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 enriched in UPEC isolates compared to 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 (ΔfecA/ΔentB) abrogates use of ferric citrate as an iron source, and fecA provides an advantage in human urine in the 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. IMPORTANCE UPEC, the primary causative agent of uncomplicated UTI, is responsible for five billion dollars in health care costs in the United States 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 UPEC isolates, underlining the importance of the continued study of these strains to fully understand their mechanisms of pathogenesis.

The ArcAB Two-Component System: Function in Metabolism, Redox Control, and Infection.

ArcAB, also known as the Arc system, is a member of the two-component system family of bacterial transcriptional regulators and is composed of sensor kinase ArcB and response regulator ArcA. In this review, we describe the structure and function of these proteins and assess the state of the literature regarding ArcAB as a sensor of oxygen consumption. The bacterial quinone pool is the primary modulator of ArcAB activity, but questions remain for how this regulation occurs. This review highlights the role of quinones and their oxidation state in activating and deactivating ArcB and compares competing models of the regulatory mechanism. The cellular processes linked to ArcAB regulation of central metabolic pathways and potential interactions of the Arc system with other regulatory systems are also reviewed. Recent evidence for the function of ArcAB under aerobic conditions is challenging the long-standing characterization of this system as strictly an anaerobic global regulator, and the support for additional ArcAB functionality in this context is explored. Lastly, ArcAB-controlled cellular processes with relevance to infection are assessed.

CpxA Phosphatase Inhibitor Activates CpxRA and Is a Potential Treatment for Uropathogenic Escherichia coli in a Murine Model of Infection.

CpxRA is an envelope stress response system that is highly conserved in the Enterobacteriaceae. CpxA has kinase activity for CpxR and phosphatase activity for phospho-CpxR (CpxR-P), a transcription factor. In response to membrane stress, CpxR-P is produced and upregulates genes involved in membrane repair and downregulates genes that encode virulence factors that are trafficked across the cell membrane. Mutants that constitutively activate CpxRA in Salmonella enterica serovar Typhimurium and in uropathogenic Escherichia coli (UPEC) are attenuated in murine models. We hypothesized that pharmacologic activation of CpxR could serve as an antimicrobial/antivirulence strategy and recently showed that 2,3,4,9-tetrahydro-1H-carbazol-1-amines activate the CpxRA system by inhibiting CpxA phosphatase activity. Here, we tested the ability of a series of three CpxRA-activating compounds with increasing potency to clear UPEC stain CFT073 in a murine urinary tract infection model. We show that these compounds are well tolerated and achieve sufficient levels to activate CpxR in the kidneys, bladder, and urine. Although the first two compounds were ineffective in promoting clearance of CFT073 in the murine model, the most potent derivative, compound 26, significantly reduced bacterial recovery in the urine and trended toward reducing bacterial recovery in the bladder and kidneys, with efficacy similar to ciprofloxacin. Treatment of CFT073 cultured in human urine with compound 26 fostered accumulation of CpxR-P and decreased the expression of proteins involved in siderophore biosynthesis and binding, heme degradation, and flagellar movement. These studies suggest that chemical activation of CpxRA may present a viable strategy for treating infections due to UPEC. IMPORTANCE The increasing prevalence of urinary tract infections (UTIs) due to antibiotic-resistant uropathogenic Escherichia coli (UPEC) is a major public health concern. Bacteria contain proteins that sense their environment and have no human homologs and, thus, are attractive drug targets. CpxRA is a conserved sensing system whose function is to reduce stress in the bacterial cell membrane; activation of CpxRA reduces the expression of virulence determinants, which must cross the cell membrane to reach the bacterial surface. We previously identified a class of compounds that activate CpxRA. We show in a mouse UTI model that our most potent compound significantly reduced recovery of UPEC in the urine, trended toward reducing bacterial recovery in the bladder and kidneys, did not kill UPEC, and downregulated multiple proteins involved in UPEC virulence. Since these compounds do not act by a killing mechanism, they have potential to treat UTIs caused by antibiotic-resistant bacteria.

Identification of distinct capsule types associated with Serratia marcescens infection isolates.

Serratia marcescens is a versatile opportunistic pathogen that can cause a variety of infections, including bacteremia. Our previous work established that the capsule polysaccharide (CPS) biosynthesis and translocation locus contributes to the survival of S. marcescens in a murine model of bacteremia and in human serum. In this study, we determined the degree of capsule genetic diversity among S. marcescens isolates. Capsule loci (KL) were extracted from >300 S. marcescens genome sequences and compared. A phylogenetic comparison of KL sequences demonstrated a substantial level of KL diversity within S. marcescens as a species and a strong delineation between KL sequences originating from infection isolates versus environmental isolates. Strains from five of the identified KL types were selected for further study and electrophoretic analysis of purified CPS indicated the production of distinct glycans. Polysaccharide composition analysis confirmed this observation and identified the constituent monosaccharides for each strain. Two predominant infection-associated clades, designated KL1 and KL2, emerged from the capsule phylogeny. Bacteremia strains from KL1 and KL2 were determined to produce ketodeoxynonulonic acid and N-acetylneuraminic acid, two sialic acids that were not found in strains from other clades. Further investigation of KL1 and KL2 sequences identified two genes, designated neuA and neuB, that were hypothesized to encode sialic acid biosynthesis functions. Disruption of neuB in a KL1 isolate resulted in the loss of sialic acid and CPS production. The absence of sialic acid and CPS production also led to increased susceptibility to internalization by a human monocytic cell line, demonstrating that S. marcescens phagocytosis resistance requires CPS. Together, these results establish the capsule genetic repertoire of S. marcescens and identify infection-associated clades with sialic acid CPS components.

Loss of an Intimin-Like Protein Encoded on a Uropathogenic E. coli Pathogenicity Island Reduces Inflammation and Affects Interactions with the Urothelium.

Uropathogenic Escherichia coli (UPEC) causes the majority of uncomplicated urinary tract infections (UTI), which affect nearly half of women worldwide. Many UPEC strains carry an annotated intimin-like adhesin (ila) locus in their genome related to a well-characterized virulence factor in diarrheagenic E. coli pathotypes. Its role in UPEC uropathogenesis, however, remains unknown. In prototype UPEC strain CFT073, there is an ila locus that contains three predicted intimin-like genes, sinH, sinI, and ratA. We used in silico approaches to determine the phylogeny and genomic distribution of this locus among uropathogens. We found that the currently annotated intimin locus-encoded proteins in CFT073 are more closely related to invasin proteins found in Salmonella. Deletion of the individual sinH, sinI, and ratA genes did not result in measurable effects on growth, biofilm formation, or motility in vitro. On average, sinH was more highly expressed in clinical strains during active human UTI than in human urine ex vivo. Unexpectedly, we found that strains lacking this ila locus had increased adherence to bladder cells in vitro, coupled with a decrease in bladder cell invasion and death. The sinH mutant displayed a significant fitness defect in the murine model of ascending UTI, including reduced inflammation in the bladder. These data confirmed an inhibitory role in bladder cell adherence to facilitate invasion and inflammation; therefore, the ila locus should be termed invasin-like rather than intimin-like. Collectively, our data suggest that loss of this locus mediates measurable interactions with bladder cells in vitro and contributes to fitness during UTI.

Replication Dynamics for Six Gram-Negative Bacterial Species during Bloodstream Infection.

Bloodstream infections (BSI) are a major public health burden due to high mortality rates and the cost of treatment. The impact of BSI is further compounded by a rise in antibiotic resistance among Gram-negative species associated with these infections. Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, Enterobacter hormaechei, Citrobacter freundii, and Acinetobacter baumannii are all common causes of BSI, which can be recapitulated in a murine model. The objective of this study was to characterize infection kinetics and bacterial replication rates during bacteremia for these six pathogens to gain a better understanding of bacterial physiology during infection. Temporal observations of bacterial burdens of the tested species demonstrated varied abilities to establish colonization in the spleen, liver, or kidney. K. pneumoniae and S. marcescens expanded rapidly in the liver and kidney, respectively. Other organisms, such as C. freundii and E. hormaechei, were steadily cleared from all three target organs throughout the infection. In situ replication rates measured by whole-genome sequencing of bacterial DNA recovered from murine spleens demonstrated that each species was capable of sustained replication at 24 h postinfection, and several species demonstrated <60-min generation times. The relatively short generation times observed in the spleen were in contrast to an overall decrease in bacterial burden for some species, suggesting that the rate of immune-mediated clearance exceeded replication. Furthermore, bacterial generation times measured in the murine spleen approximated those measured during growth in human serum cultures. Together, these findings provide insight into the infection kinetics of six medically important species during bacteremia. IMPORTANCE Bloodstream infections are a global public health problem. The goal of this work was to determine the replication characteristics of Gram-negative bacterial species in the host following bloodstream infection. The number of bacteria in major organs is likely determined by a balance between replication rates and the ability of the host to clear bacteria. We selected a cohort of six species from three families that represent common causative agents of bloodstream infections in humans and determined their replication rates in a murine bacteremia model. We found that the bacteria grow rapidly in the spleen, demonstrating that they can obtain the necessary nutrients for growth in this environment. However, the overall number of bacteria decreased in most cases, suggesting that killing of bacteria outpaces their growth. Through a better understanding of how bacteria replicate during bloodstream infections, we aim to gain insight into future means of combating these infections.

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 hindered 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 effect on CPS biosynthesis and hmv, were evaluated for their impact on CPS thickness, serum resistance, host cell association, and fitness in a murine model of disseminating pneumonia. Altogether, these data demonstrate that hmv requires both CPS biosynthesis and other cellular factors, and that hmv and CPS may serve distinct functions during pathogenesis. The integration of hmv and CPS to the metabolic status of the cell suggests that hvKp may require certain nutrients to specifically cause deep tissue infections.

Pathogenesis of Gram-Negative Bacteremia.

Gram-negative bacteremia is a devastating public health threat, with high mortality in vulnerable populations and significant costs to the global economy. Concerningly, rates of both Gram-negative bacteremia and antimicrobial resistance in the causative species are increasing. Gram-negative bacteremia develops in three phases. First, bacteria invade or colonize initial sites of infection. Second, bacteria overcome host barriers, such as immune responses, and disseminate from initial body sites to the bloodstream. Third, bacteria adapt to survive in the blood and blood-filtering organs. To develop new therapies, it is critical to define species-specific and multispecies fitness factors required for bacteremia in model systems that are relevant to human infection. A small subset of species is responsible for the majority of Gram-negative bacteremia cases, including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii The few bacteremia fitness factors identified in these prominent Gram-negative species demonstrate shared and unique pathogenic mechanisms at each phase of bacteremia progression. Capsule production, adhesins, and metabolic flexibility are common mediators, whereas only some species utilize toxins. This review provides an overview of Gram-negative bacteremia, compares animal models for bacteremia, and discusses prevalent Gram-negative bacteremia species.

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, including a well-established mouse model of UTI. However, the extent to which bacterial physiologies differ between experimental models and human infections remains largely understudied. To address this important issue, we compared the transcriptomes of three different UPEC isolates in human infection and under 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 coefficients of 0.86 to 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) downregulated in patients. Importantly, gene expression levels 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 hinted 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.IMPORTANCE Different experimental models have been used to study UPEC pathogenesis, including in vitro cultures in different media, tissue culture, and mouse models of infection. The last 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 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.