Michael A. Bachman

Klebsiella pneumoniae yersiniabactin promotes respiratory tract infection through evasion of lipocalin 2.

ABSTRACT Klebsiella pneumoniae is a pathogen of increasing concern because of multidrug resistance, especially due to K. pneumoniae carbapenemases (KPCs). K. pneumoniae must acquire iron to replicate, and it utilizes iron-scavenging siderophores, such as enterobactin (Ent). The innate immune protein lipocalin 2 (Lcn2) is able to specifically bind Ent and disrupt iron acquisition. To determine whether K. pneumoniae must produce Lcn2-resistant siderophores to cause disease, we examined siderophore production by clinical isolates (n = 129) from respiratory, urine, blood, and stool samples and by defined siderophore mutants through genotyping and liquid chromatography-mass spectrometry. Three categories of K. pneumoniae isolates were identified: enterobactin positive (Ent+) (81%), enterobactin and yersiniabactin positive (Ent+ Ybt+) (17%), and enterobactin and salmochelin (glycosylated Ent) positive (Ent+ gly-Ent+) with or without Ybt (2%). Ent+ Ybt+ strains were significantly overrepresented among respiratory tract isolates (P = 0.0068) and β-lactam-resistant isolates (P = 0.0019), including the epidemic KPC-producing clone multilocus sequence type 258 (ST258). In ex vivo growth assays, gly-Ent but not Ybt allowed evasion of Lcn2 in human serum, whereas siderophores were dispensable for growth in human urine. In a murine pneumonia model, an Ent+ strain was an opportunistic pathogen that was completely inhibited by Lcn2 but caused severe, disseminated disease in Lcn2 −/− mice. In contrast, an Ent+ Ybt+ strain was a frank respiratory pathogen, causing pneumonia despite Lcn2. However, Lcn2 retained partial protection against disseminated disease. In summary, Ybt is a virulence factor that is prevalent among KPC-producing K. pneumoniae isolates and promotes respiratory tract infections through evasion of Lcn2.

Mucosal Lipocalin 2 Has Pro-Inflammatory and Iron-Sequestering Effects in Response to Bacterial Enterobactin

Nasal colonization by both gram-positive and gram-negative pathogens induces expression of the innate immune protein lipocalin 2 (Lcn2). Lcn2 binds and sequesters the iron-scavenging siderophore enterobactin (Ent), preventing bacterial iron acquisition. In addition, Lcn2 bound to Ent induces release of IL-8 from cultured respiratory cells. As a countermeasure, pathogens of the Enterobacteriaceae family such as Klebsiella pneumoniae produce additional siderophores such as yersiniabactin (Ybt) and contain the iroA locus encoding an Ent glycosylase that prevents Lcn2 binding. Whereas the ability of Lcn2 to sequester iron is well described, the ability of Lcn2 to induce inflammation during infection is unknown. To study each potential effect of Lcn2 on colonization, we exploited K. pneumoniae mutants that are predicted to be susceptible to Lcn2-mediated iron sequestration (iroA ybtS mutant) or inflammation (iroA mutant), or to not interact with Lcn2 (entB mutant). During murine nasal colonization, the iroA ybtS double mutant was inhibited in an Lcn2-dependent manner, indicating that the iroA locus protects against Lcn2-mediated growth inhibition. Since the iroA single mutant was not inhibited, production of Ybt circumvents the iron sequestration effect of Lcn2 binding to Ent. However, colonization with the iroA mutant induced an increased influx of neutrophils compared to the entB mutant. This enhanced neutrophil response to Ent-producing K. pneumoniae was Lcn2-dependent. These findings suggest that Lcn2 has both pro-inflammatory and iron-sequestering effects along the respiratory mucosa in response to bacterial Ent. Therefore, Lcn2 may represent a novel mechanism of sensing microbial metabolism to modulate the host response appropriately.

A tool kit for quantifying eukaryotic rRNA gene sequences from human microbiome samples

Eukaryotic microorganisms are important but understudied components of the human microbiome. Here we present a pipeline for analysis of deep sequencing data on single cell eukaryotes. We designed a new 18S rRNA gene-specific PCR primer set and compared a published rRNA gene internal transcribed spacer (ITS) gene primer set. Amplicons were tested against 24 specimens from defined eukaryotes and eight well-characterized human stool samples. A software pipeline https://sourceforge.net/projects/brocc/ was developed for taxonomic attribution, validated against simulated data, and tested on pyrosequence data. This study provides a well-characterized tool kit for sequence-based enumeration of eukaryotic organisms in human microbiome samples.

Genetic Evidence that Legionella pneumophila RpoS Modulates Expression of the Transmission Phenotype in Both the Exponential Phase and the Stationary Phase

ABSTRACT The opportunistic pathogen Legionella pneumophila alternates between two states: replication within phagocytes and transmission between host amoebae or macrophages. In broth cultures that model this life cycle, during the replication period, CsrA inhibits expression of transmission traits. When nutrients become limiting, the alarmone (p)ppGpp accumulates and the sigma factors RpoS and FliA and the positive activators LetA/S and LetE promote differentiation to the transmissible form. Here we show that when cells enter the postexponential growth phase, RpoS increases expression of the transmission genes fliA, flaA, and mip, factors L. pneumophila needs to establish a new replication niche. In contrast, in exponential (E)-phase cells whose (p)ppGpp levels are low, rpoS inhibits expression of transmission traits, on the basis of three separate observations. First, rpoS RNA levels peak in the E phase, suggestive of a role for RpoS during replication. Second, in multiple copies, rpoS decreases the amounts of csrA, letE, fliA, and flaA transcripts and inhibits the transmission traits of motility, infectivity, and cytotoxicity. Third, rpoS blocks expression of cytotoxicity and motility by E-phase bacteria that have been induced to express the LetA activator ectopically. The data are discussed in the context of a model in which the alarmone (p)ppGpp enables RpoS to outcompete other sigma factors for binding to RNA polymerase to promote transcription of transmission genes, while LetA/S acts in parallel to relieve CsrA posttranscriptional repression of the transmission regulon. By coupling transcriptional and posttranscriptional control pathways, intracellular L. pneumophila could respond to stress by rapidly differentiating to a transmissible form.

Interaction of Lipocalin 2, Transferrin, and Siderophores Determines the Replicative Niche of Klebsiella pneumoniae during Pneumonia

ABSTRACT Pathogenic bacteria require iron for replication within their host. Klebsiella pneumoniae and other Gram-negative pathogens produce the prototypical siderophore enterobactin (Ent) to scavenge iron in vivo. In response, mucosal surfaces secrete lipocalin 2 (Lcn2), an innate immune protein that binds Ent to disrupt bacterial iron acquisition and promote acute inflammation during colonization. A subset of K. pneumoniae isolates attempt to evade Lcn2 by producing glycosylated Ent (Gly-Ent, salmochelin) or the alternative siderophore yersiniabactin (Ybt). However, these siderophores are not functionally equivalent and differ in their abilities to promote growth in the upper respiratory tract, lungs, and serum. To understand how Lcn2 exploits functional differences between siderophores, isogenic mutants of an Ent+ Gly-Ent+ Ybt+ K. pneumoniae strain were inoculated into Lcn2+/+ and Lcn2−/− mice, and the pattern of pneumonia was examined. Lcn2 effectively protected against the iroA ybtS mutant (Ent+ Gly-Ent− Ybt−). Lcn2+/+ mice had small foci of pneumonia, whereas Lcn2−/− mice had many bacteria in the perivascular space. The entB mutant (Ent− Ybt+ Gly-Ent−) caused moderate bronchopneumonia but did not invade the transferrin-containing perivascular space. Accordingly, transferrin blocked Ybt-dependent growth in vitro. The wild type and the iroA mutant, which both produce Ent and Ybt, had a mixed phenotype, causing a moderate bronchopneumonia in Lcn2+/+ mice and perivascular overgrowth in Lcn2−/− mice. Together, these data indicate that Lcn2, in combination with transferrin, confines K. pneumoniae to the airways and prevents invasion into tissue containing the pulmonary vasculature. IMPORTANCE Gram-negative bacteria are a common cause of severe hospital-acquired infections. To cause disease, they must obtain iron and secrete the small molecule enterobactin to do so. Animal models of pneumonia using Klebsiella pneumoniae indicate that enterobactin promotes severe disease. Accordingly, the host defense protein lipocalin 2 exploits this common target by binding enterobactin and disrupting its function. However, pathogenic bacteria often make additional siderophores that lipocalin 2 cannot bind, such as yersiniabactin, which could make this host defense ineffective. This work compares the pattern and severity of pneumonia caused by K. pneumoniae based on which siderophores it produces. The results indicate that enterobactin promotes growth around blood vessels that are rich in the iron-binding protein transferrin, but yersiniabactin does not. Together, transferrin and lipocalin 2 protect this space against all types of K. pneumoniae tested. Therefore, the ability to acquire iron determines where bacteria can grow in the lung. Gram-negative bacteria are a common cause of severe hospital-acquired infections. To cause disease, they must obtain iron and secrete the small molecule enterobactin to do so. Animal models of pneumonia using Klebsiella pneumoniae indicate that enterobactin promotes severe disease. Accordingly, the host defense protein lipocalin 2 exploits this common target by binding enterobactin and disrupting its function. However, pathogenic bacteria often make additional siderophores that lipocalin 2 cannot bind, such as yersiniabactin, which could make this host defense ineffective. This work compares the pattern and severity of pneumonia caused by K. pneumoniae based on which siderophores it produces. The results indicate that enterobactin promotes growth around blood vessels that are rich in the iron-binding protein transferrin, but yersiniabactin does not. Together, transferrin and lipocalin 2 protect this space against all types of K. pneumoniae tested. Therefore, the ability to acquire iron determines where bacteria can grow in the lung.

The LetE Protein Enhances Expression of Multiple LetA/LetS-Dependent Transmission Traits by Legionella pneumophila

ABSTRACT Legionella pneumophila colonizes freshwater amoebae and can also replicate within alveolar macrophages. When their nutrient supply is exhausted, replicating bacteria become cytotoxic, motile, and infectious, which is thought to promote transmission to a new amoeba. The differentiation of L. pneumophila is coordinated by the sigma factors RpoS and FliA and the two-component regulator LetA/LetS and is enhanced by the letE locus. Here we demonstrate that letE promotes motility by increasing expression of the flagellin gene flaA but has little impact on the transcription of fliA, the flagellar sigma factor gene. In addition to promoting motility, letE induces the characteristic shape, pigment, and heat resistance of stationary-phase L. pneumophila. To gain insight into how letE promotes the expression of the transmission phenotype, we designed molecular genetic experiments to discriminate between the following three models: letE mutations are polar on milX; letE encodes a small novel protein; or, by analogy to csrB, letE encodes a regulatory RNA that sequesters CsrA to relieve repression. We report that letE encodes an activator protein, as it does not complement an Escherichia coli csrB mutant, it directs the synthesis of an ∼12-kDa polypeptide, and a letE nonsense mutation eliminates function. A monocistronic letE RNA is abundant during the exponential phase, and its decay during the stationary phase requires RpoS and LetA/LetS. We also discuss how the LetE protein may interact with LetA/LetS and CsrA to enhance L. pneumophila differentiation to a transmissible form.

Genome-Wide Identification of Klebsiella pneumoniae Fitness Genes during Lung Infection

ABSTRACT Klebsiella pneumoniae is an urgent public health threat because of resistance to carbapenems, antibiotics of last resort against Gram-negative bacterial infections. Despite the fact that K. pneumoniae is a leading cause of pneumonia in hospitalized patients, the bacterial factors required to cause disease are poorly understood. Insertion site sequencing combines transposon mutagenesis with high-throughput sequencing to simultaneously screen thousands of insertion mutants for fitness defects during infection. Using the recently sequenced K. pneumoniae strain KPPR1 in a well-established mouse model of pneumonia, insertion site sequencing was performed on a pool of >25,000 transposon mutants. The relative fitness requirement of each gene was ranked based on the ratio of lung to inoculum read counts and concordance between insertions in the same gene. This analysis revealed over 300 mutants with at least a 2-fold fitness defect and 69 with defects ranging from 10- to >2,000-fold. Construction of 6 isogenic mutants for use in competitive infections with the wild type confirmed their requirement for lung fitness. Critical fitness genes included those for the synthesis of branched-chain and aromatic amino acids that are essential in mice and humans, the transcriptional elongation factor RfaH, and the copper efflux pump CopA. The majority of fitness genes were conserved among reference strains representative of diverse pathotypes. These results indicate that regulation of outer membrane components and synthesis of amino acids that are essential to its host are critical for K. pneumoniae fitness in the lung. IMPORTANCE Klebsiella pneumoniae is a bacterium that commonly causes pneumonia in patients after they are admitted to the hospital. K. pneumoniae is becoming resistant to all available antibiotics, and when these infections spread to the bloodstream, over half of patients die. Since currently available antibiotics are failing, we must discover new ways to treat these infections. In this study, we asked what genes the bacterium needs to cause an infection, since the proteins encoded by these genes could be targets for new antibiotics. We identified over 300 genes that K. pneumoniae requires to grow in a mouse model of pneumonia. Many of the genes that we identified are found in K. pneumoniae isolates from throughout the world, including antibiotic-resistant forms. If new antibiotics could be made against the proteins that these genes encode, they may be broadly effective against K. pneumoniae. Klebsiella pneumoniae is a bacterium that commonly causes pneumonia in patients after they are admitted to the hospital. K. pneumoniae is becoming resistant to all available antibiotics, and when these infections spread to the bloodstream, over half of patients die. Since currently available antibiotics are failing, we must discover new ways to treat these infections. In this study, we asked what genes the bacterium needs to cause an infection, since the proteins encoded by these genes could be targets for new antibiotics. We identified over 300 genes that K. pneumoniae requires to grow in a mouse model of pneumonia. Many of the genes that we identified are found in K. pneumoniae isolates from throughout the world, including antibiotic-resistant forms. If new antibiotics could be made against the proteins that these genes encode, they may be broadly effective against K. pneumoniae.

Molecular Epidemiology of Colonizing and Infecting Isolates of Klebsiella pneumoniae

K. pneumoniae commonly infects hospitalized patients, and these infections are increasingly resistant to carbapenems, the antibiotics of last resort for life-threatening bacterial infections. To prevent and treat these infections, we must better understand how K. pneumoniae causes disease and discover new ways to predict and detect infections. This study demonstrates that colonization with K. pneumoniae in the intestinal tract is strongly linked to subsequent infection. This finding helps to identify a potential time frame and possible approach for intervention: the colonizing strain from a patient could be isolated as part of a risk assessment, and antibiotic susceptibility testing could guide empirical therapy if the patient becomes acutely ill. ABSTRACT Klebsiella pneumoniae is among the most common causes of hospital-acquired infections and has emerged as an urgent threat to public health due to carbapenem antimicrobial resistance. K. pneumoniae commonly colonizes hospitalized patients and causes extraintestinal infections such as urinary tract infection, bloodstream infection (septicemia), and pneumonia. If colonization is an intermediate step before infection, then detection and characterization of colonizing isolates could enable strategies to prevent or empirically treat K. pneumoniae infections in hospitalized patients. However, the strength of the association between colonization and infection is unclear. To test the hypothesis that hospitalized patients become infected with their colonizing strain, 1,765 patients were screened for rectal colonization with K. pneumoniae, and extraintestinal isolates from these same patients were collected over a 3-month period in a cohort study design. The overall colonization prevalence was 23.0%. After adjustment for other patient factors, colonization was significantly associated with subsequent infection: 21 of 406 (5.2%) colonized patients later had extraintestinal infection, compared to 18 of 1,359 (1.3%) noncolonized patients (adjusted odds ratio [OR], 4.01; 95% confidence interval, 2.08 to 7.73; P < 0.001). Despite a high diversity of colonizing isolates, 7/7 respiratory, 4/4 urinary, and 2/5 bloodstream isolates from colonized patients matched the patient corresponding rectal swab isolates, based on wzi capsular typing, multilocus sequence typing (MLST), and whole-genome sequence analysis. These results suggest that K. pneumoniae colonization is directly associated with progression to extraintestinal infection. IMPORTANCE K. pneumoniae commonly infects hospitalized patients, and these infections are increasingly resistant to carbapenems, the antibiotics of last resort for life-threatening bacterial infections. To prevent and treat these infections, we must better understand how K. pneumoniae causes disease and discover new ways to predict and detect infections. This study demonstrates that colonization with K. pneumoniae in the intestinal tract is strongly linked to subsequent infection. This finding helps to identify a potential time frame and possible approach for intervention: the colonizing strain from a patient could be isolated as part of a risk assessment, and antibiotic susceptibility testing could guide empirical therapy if the patient becomes acutely ill.

Klebsiella pneumoniae Siderophores Induce Inflammation, Bacterial Dissemination, and HIF-1α Stabilization during Pneumonia

ABSTRACT Klebsiella pneumoniae is a Gram-negative pathogen responsible for a wide range of infections, including pneumonia and bacteremia, and is rapidly acquiring antibiotic resistance. K. pneumoniae requires secretion of siderophores, low-molecular-weight, high-affinity iron chelators, for bacterial replication and full virulence. The specific combination of siderophores secreted by K. pneumoniae during infection can impact tissue localization, systemic dissemination, and host survival. However, the effect of these potent iron chelators on the host during infection is unknown. In vitro, siderophores deplete epithelial cell iron, induce cytokine secretion, and activate the master transcription factor hypoxia inducible factor-1α (HIF-1α) protein that controls vascular permeability and inflammatory gene expression. Therefore, we hypothesized that siderophore secretion by K. pneumoniae directly contributes to inflammation and bacterial dissemination during pneumonia. To examine the effects of siderophore secretion independently of bacterial growth, we performed infections with tonB mutants that persist in vivo but are deficient in siderophore import. Using a murine model of pneumonia, we found that siderophore secretion by K. pneumoniae induces the secretion of interleukin-6 (IL-6), CXCL1, and CXCL2, as well as bacterial dissemination to the spleen, compared to siderophore-negative mutants at an equivalent bacterial number. Furthermore, we determined that siderophore-secreting K. pneumoniae stabilized HIF-1α in vivo and that bacterial dissemination to the spleen required alveolar epithelial HIF-1α. Our results indicate that siderophores act directly on the host to induce inflammatory cytokines and bacterial dissemination and that HIF-1α is a susceptibility factor for bacterial invasion during pneumonia. IMPORTANCE Klebsiella pneumoniae causes a wide range of bacterial diseases, including pneumonia, urinary tract infections, and sepsis. To cause infection, K. pneumoniae steals iron from its host by secreting siderophores, small iron-chelating molecules. Classically, siderophores are thought to worsen infections by promoting bacterial growth. In this study, we determined that siderophore-secreting K. pneumoniae causes lung inflammation and bacterial dissemination to the bloodstream independently of bacterial growth. Furthermore, we determined that siderophore-secreting K. pneumoniae activates a host protein, hypoxia inducible factor (HIF)-1α, and requires it for siderophore-dependent bacterial dissemination. Although HIF-1α can protect against some infections, it appears to worsen infection with K. pneumoniae. Together, these results indicate that bacterial siderophores directly alter the host response to pneumonia in addition to providing iron for bacterial growth. Therapies that disrupt production of siderophores could provide a two-pronged attack against K. pneumoniae infection by preventing bacterial growth and preventing bacterial dissemination to the blood. Klebsiella pneumoniae causes a wide range of bacterial diseases, including pneumonia, urinary tract infections, and sepsis. To cause infection, K. pneumoniae steals iron from its host by secreting siderophores, small iron-chelating molecules. Classically, siderophores are thought to worsen infections by promoting bacterial growth. In this study, we determined that siderophore-secreting K. pneumoniae causes lung inflammation and bacterial dissemination to the bloodstream independently of bacterial growth. Furthermore, we determined that siderophore-secreting K. pneumoniae activates a host protein, hypoxia inducible factor (HIF)-1α, and requires it for siderophore-dependent bacterial dissemination. Although HIF-1α can protect against some infections, it appears to worsen infection with K. pneumoniae. Together, these results indicate that bacterial siderophores directly alter the host response to pneumonia in addition to providing iron for bacterial growth. Therapies that disrupt production of siderophores could provide a two-pronged attack against K. pneumoniae infection by preventing bacterial growth and preventing bacterial dissemination to the blood.

Bacterial Siderophores That Evade or Overwhelm Lipocalin 2 Induce Hypoxia Inducible Factor 1α and Proinflammatory Cytokine Secretion in Cultured Respiratory Epithelial Cells

ABSTRACT Iron is essential for many cellular processes and is required by bacteria for replication. To acquire iron from the host, pathogenic Gram-negative bacteria secrete siderophores, including enterobactin (Ent). However, Ent is bound by the host protein lipocalin 2 (Lcn2), preventing bacterial reuptake of aferric or ferric Ent. Furthermore, the combination of Ent and Lcn2 (Ent+Lcn2) leads to enhanced secretion of interleukin-8 (IL-8) compared to that induced by either stimulus alone. Modified or structurally distinct siderophores, including yersiniabactin (Ybt) and glycosylated Ent (GlyEnt, or salmochelin), deliver iron to bacteria despite the presence of Lcn2. We hypothesized that the robust immune response to Ent and Lcn2 requires iron chelation rather than the Ent+Lcn2 complex itself and also can be stimulated by Lcn2-evasive siderophores. To test this hypothesis, cultured respiratory epithelial cells were stimulated with combinations of purified siderophores and Lcn2 and analyzed by gene expression microarrays, quantitative PCR, and cytokine immunoassays. Ent caused HIF-1α protein stabilization, induced the expression of genes regulated by hypoxia-inducible factor 1α (HIF-1α), and repressed genes involved in cell cycle and DNA replication, whereas Lcn2 induced expression of proinflammatory cytokines. Iron chelation by excess Ent or Ybt significantly increased Lcn2-induced secretion of IL-8, IL-6, and CCL20. Stabilization of HIF-1α was sufficient to enhance Lcn2-induced IL-6 secretion. These data indicate that respiratory epithelial cells can respond to bacterial siderophores that evade or overwhelm Lcn2 binding by increasing proinflammatory cytokine production.