Bloodstream infections: mechanisms of pathogenesis and opportunities for intervention.

Bloodstream infections (BSIs) are common in hospitals, often life-threatening and increasing in prevalence. Microorganisms in the blood are usually rapidly cleared by the immune system and filtering organs but, in some cases, they can cause an acute infection and trigger sepsis, a systemic response to infection that leads to circulatory collapse, multiorgan dysfunction and death. Most BSIs are caused by bacteria, although fungi also contribute to a substantial portion of cases. Escherichia coli, Staphylococcus aureus, coagulase-negative Staphylococcus, Klebsiella pneumoniae and Candida albicans are leading causes of BSIs, although their prevalence depends on patient demographics and geographical region. Each species is equipped with unique factors that aid in the colonization of initial sites and dissemination and survival in the blood, and these factors represent potential opportunities for interventions. As many pathogens become increasingly resistant to antimicrobials, new approaches to diagnose and treat BSIs at all stages of infection are urgently needed. In this Review, we explore the prevalence of major BSI pathogens, prominent mechanisms of BSI pathogenesis, opportunities for prevention and diagnosis, and treatment options.

Asymptomatic bacteriuria screening for developing countries using a modified water quality test kit.

Between 2% and 15% of pregnant women unknowingly experience asymptomatic bacteriuria (ASB), defined as ≥105 CFU per milliliter of urine in the absence of symptoms. ASB increases the risk of adverse pregnancy outcomes including pyelonephritis, preterm labor, and low-birth weight infants. While pregnant women in the United States are routinely screened for ASB, those in developing countries with limited resources and funding lack an accurate mechanism for ASB screening. Aquagenx water quality test kits detect Escherichia coli, the most common causative agent of ASB, and total coliform bacteria in drinking water via colorimetric and fluorescent indicators. We found that the Aquagenx system is compatible with human urine and then proceeded to develop an ASB screening protocol using disposable inoculating loops. Our protocol diagnosed artificial ASB- samples (104 CFU/mL E. coli) with a false positive (FP) rate of 33% (n = 18) and ASB+ (105 CFU/mL E. coli) with a false negative (FN) rate of 5.6% (n = 18). Clinical sample testing with our protocol revealed a FP rate of 0% in ASB- samples (n = 28) and a FN rate of 0% in ASB+ samples caused by coliforms (n = 13). Aquagenx did not detect ASB in nine clinical samples with non-coliform etiological agents due to the limitations of the technology. However, with very high accuracy for detection of E. coli and other causative agents that collectively account for 90.1% of ASB cases, these kits could be used as a diagnostic ASB screening tool in developing countries in which there is currently no alternative to urine culture.IMPORTANCEAsymptomatic bacteriuria (ASB) affects 2%-15% of pregnant women and can result in adverse maternal and fetal outcomes if left undetected and untreated. In the United States and other developed nations, pregnant women are regularly screened for ASB via urine culture. However, in low-resource countries where bacterial culture is not available, dipstick testing is used. Although accurate in cases of symptomatic bacteriuria, dipstick detection is ineffective for detecting ASB. Here, we made use of an existing water quality field test for ASB urine screening, which would be readily deployable in low-resource settings. We optimized a dilution protocol for sampling patient urine within the detection limits of the Aquagenx kit technology. Overall, we were able to detect ASB samples with Gram-negative pathogens that collectively account for 90% of all ASB cases. Utilization of this repurposed technology for proactive medical screening may help prevent adverse pregnancy and birth outcomes due to ASB.

Development of a suite of Proteus mirabilis-derived urea-inducible promoters.

Catheter-associated urinary tract infections (CAUTIs) are a significant burden on healthcare systems, accounting for up to 40% of hospital-acquired infections globally. A prevalent CAUTI pathogen, Proteus mirabilis, is an understudied Gram-negative bacterium. One sequela of P. mirabilis CAUTI is the production of urinary stones, which complicates treatment and clearing of the infection. Stone formation is induced by the activity of urease, a nickel-metalloenzyme that is regulated by UreR in a urea-dependent manner. As urea is abundant in the urinary tract, urease genes are highly expressed during experimental UTI. We sought to leverage the urease promoter to create an expression system that would enable urea-inducible expression of genes during in vitro experiments as well as during experimental UTI. During preliminary studies, we observed unexpectedly high levels of basal expression of the urease promoter. This was somewhat dependent on the presence of regulator UreR. To further develop this expression system, we generated a series of reporter constructs to assess the impact of specific promoter elements on promoter activity in the presence and absence of urea. Elements of interest included known regulatory binding sites, alternative translational start sites, and single-nucleotide polymorphisms identified through comparative genomics. This work describes a suite of urea-inducible promoters, constructed during this study, that exhibit a variety of expression dynamics, providing a customizable platform for gene expression.IMPORTANCEUrea is an inexpensive molecule that can easily be supplied during in vitro experiments. A urea-inducible promoter would also be activated by environments where urea naturally occurs, such as in the urinary tract. Thus, the development of a urea-inducible system for selective gene expression is of great interest to the field of uropathogenesis as it would enable selective gene induction during experimental urinary tract infection. This expression system would also have important applications for recombinant protein production in biotech and manufacturing.

Infection characteristics among Serratia marcescens capsule lineages.

Serratia marcescens is a healthcare-associated pathogen that causes bloodstream infections, pneumonia, and urinary tract infections. The capsule polysaccharide of S. marcescens is a critical fitness determinant during infection and recent work established the relationship between capsule locus (KL) genetic sequences within the species. Strains belonging to KL1 and KL2 capsule clades produce sialylated polysaccharides and represent the largest subpopulation of isolates from clinical origin while the S. marcescens type strain and other environmental isolates were classified as KL5. In this work, the contribution of these and other capsules to pathogenesis in multiple infection models was determined. Using a murine tail vein injection model of bacteremia, clinical strains demonstrated capsule-dependent colonization of spleen, liver, and kidney following inoculation. The KL5 strain, in contrast, exhibited no loss of survival in this model when capsule genes were deleted. Furthermore, the wild-type KL5 strain was cleared more rapidly from both the spleen and liver compared to a KL1 strain. Similar results were observed in 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 other organs. Finally, strains from each KL clade were tested for the role of capsule in internalization by bone marrow-derived macrophages. Only the sialylated KL1 and KL2 clade strains, representing the majority of clinical isolates, exhibited capsule-dependent inhibition of internalization, suggesting that capsule-mediated resistance to macrophage phagocytosis may enhance survival and antibacterial defenses during infection.

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 unregulated immune responses 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. Mutants were constructed for all genes in all five species. Each mutant was used to cochallenge C57BL/6 mice via tail vein injection along with each respective wild-type strain to determine competitive indices for each fitness gene. Five fitness factor genes, when mutated, attenuated mutants in four or five species in the spleen and liver (tatC, ruvA, gmhB, wzxE, arcA). Five additional fitness factor genes or operons were validated as outcompeted by wild-type in three, four, or five bacterial species in the spleen (xerC, prc, apaGH, atpG, 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.

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.

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.

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.

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.

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.

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.