Hypervirulent K. Pneumoniae Secretes More and More Active Iron-Acquisition Molecules than “Classical” K. Pneumoniae Thereby Enhancing its Virulence

A new hypervirulent (hypermucoviscous) clinical variant of Klebsiella pneumoniae (hvKP) has emerged over the last decade. Our goal is to identify new mechanisms, which increase the virulence hvKP compared to "classic" K. pneumoniae (cKP).Various growth assays were performed in human ascites, human serum, and laboratory medium with the hvKP strain hvKP1 (wt), randomly chosen blood isolates of cKP strains (cKP1-4), and mutant constructs deficient in the secretion of selected compounds. An in vivo mouse model that mimics infection due to hvKP and a quantitative siderophore assay were also used. It was established that a molecule(s)/factor(s) was secreted by hvKP1 significantly enhanced its growth and/or survival in human ascites. This molecule(s)/factor(s) also increased the growth and/or survival of hvKP1 in serum ex vivo and in an in vivo mouse model that measures metastatic spread after subcutaneous challenge, thereby further establishing biologic significance. Although features such as a size of <3kD, heat stability, and growth characteristics in ascites suggested this molecule(s) was a quorum-sensing compound, data presented demonstrates that this molecule(s)/factor(s) is involved in iron uptake and is likely a siderophore(s) or another iron-acquisition molecule. Although it is known that iron acquisition is critical for virulence, a novel aspect of this observation is that hvKP1 produces quantitatively more siderophores that appear to be biologically more active (increased affinity for iron or more resistant to host factors) than those produced by cKP strains.The data presented delineates a new mechanism by which hvKP increases its pathogenic potential compared to cKP strains. This paradigm may be broadly applicable to other extraintestinal gram-negative bacilli

IroN Functions as a Siderophore Receptor and Is a Urovirulence Factor in an Extraintestinal Pathogenic Isolate of Escherichia coli

IroN was recently identified in the extracellular pathogenic Escherichia coli strain CP9. In this study experimental evidence demonstrating that IroN mediates utilization of the siderophore enterobactin was obtained, thereby establishing IroN as a catecholate siderophore receptor. In a mouse model of ascending urinary tract infection the presence of iroN contributed significantly to CP9's ability to colonize the mouse bladder, kidneys, and urine, evidence that IroN is a urovirulence factor. However, growth in human urine ex vivo and adherence to uroepithelial cells in vitro were equivalent for an isogenic mutant deficient in IroN (CP82) and its wild-type parent (CP9). Taken together, these findings establish that IroN is a siderophore receptor and a urovirulence factor. However, uncertainty exists as to the mechanism(s) via which IroN contributes to urovirulence

The Siderophore Receptor IroN of Extraintestinal Pathogenic Escherichia coli Is a Potential Vaccine Candidate

It would be medically and economically desirable to prevent the millions of annual extraintestinal infections and the thousands of associated deaths due to Escherichia coli. Outer membrane proteins are potential vaccine candidates for the prevention of these infections. This study tested the hypotheses that the siderophore receptor IroN is antigenic and that an IroN-specific antibody response confers protection in vivo. Subcutaneous immunization with denatured IroN resulted in a significant IroN immunoglobulin G (IgG)-specific response in serum (P < 0.0001) but not a systemic or mucosal IroN-specific IgA response. In a mouse model of ascending urinary tract infection, subcutaneous immunization with denatured IroN conferred significant protection against renal (P = 0.0135 and 0.0095 in two independent experiments), but not bladder, infection. These data, together with the previously demonstrated role of IroN in virulence, its expression in human biologic fluids, and its prevalence among extraintestinal pathogenic E. coli strains, support further studies on the role of IroN as a vaccine candidate

Rat Pneumonia and Soft-Tissue Infection Models for the Study of Acinetobacter baumannii Biology▿

Acinetobacter baumannii is a bacterial pathogen of increasing medical importance. Little is known about its mechanisms of pathogenesis, and safe reliable agents with predictable activity against A. baumannii are presently nonexistent. The availability of relevant animal infection models will facilitate the study of Acinetobacter biology. In this report we tested the hypothesis that the rat pneumonia and soft-tissue infection models that our laboratory had previously used for studies of extraintestinal pathogenic Escherichia coli were clinically relevant for A. baumannii. Advantages of these models over previously described models were that the animals were not rendered neutropenic and they did not receive porcine mucin with bacterial challenge. Using the A. baumannii model pathogen 307-0294 as the challenge pathogen, the pneumonia model demonstrated all of the features of infection that are critical for a clinically relevant model: namely, bacterial growth/clearance, an ensuing host inflammatory response, acute lung injury, and, following progressive bacterial proliferation, death due to respiratory failure. We were also able to demonstrate growth of 307-0294 in the soft-tissue infection model. Next we tested the hypothesis that the soft-tissue infection model could be used to discriminate between the inherent differences in virulence of various A. baumannii clinical isolates. The ability of A. baumannii to grow and/or be cleared in this model was dependent on the challenge strain. We also hypothesized that complement is an important host factor in protecting against A. baumannii infection in vivo. In support of this hypothesis was the observation that the serum sensitivity of various A. baumannii clinical isolates in vitro roughly paralleled their growth/clearance in the soft-tissue infection model in vivo. Lastly we hypothesized that the soft-tissue infection model would serve as an efficient screening mechanism for identifying gene essentiality for drug discovery. Random mutants of 307-0294 were initially screened for lack of growth in human ascites in vitro. Selected mutants were subsequently used for challenge in the soft-tissue infection model to determine if the disrupted gene was essential for growth in vivo. Using this approach, we have been able to successfully identify a number of genes essential for the growth of 307-0294 in vivo. In summary, these models are clinically relevant and can be used to study the innate virulence of various Acinetobacter clinical isolates and to assess potential virulence factors, vaccine candidates, and drug targets in vivo and can be used for pharmacokinetic and chemotherapeutic investigations

The K1 Capsular Polysaccharide of Acinetobacter baumannii Strain 307-0294 Is a Major Virulence Factor ▿

Acinetobacter baumannii is a pathogen of increasing medical importance with a propensity to be multidrug resistant, thereby making treatment challenging. Little is known of virulence traits in A. baumannii. To identify virulence factors and potential drug targets, random transposon (Tn) mutants derived from the A. baumannii strain AB307-0294 were screened to identify genes essential for growth in human ascites fluid in vitro, an inflammatory exudative fluid. These studies led to the identification of two genes that were predicted to be required for capsule polymerization and assembly. The first, ptk, encodes a putative protein tyrosine kinase (PTK), and the second, epsA, encodes a putative polysaccharide export outer membrane protein (EpsA). Monoclonal antibodies used in flow cytometric and Western analyses confirmed that these genes are required for a capsule-positive phenotype. A capsule-positive phenotype significantly optimized growth in human ascites fluid, survival in human serum, and survival in a rat soft tissue infection model. Importantly, the clearance of the capsule-minus mutants AB307.30 (ptk mutant, capsule minus) and AB307.45 (epsA mutant, capsule minus) was complete and durable. These data demonstrated that the K1 capsule from AB307-0294 was an important protectin. Further, these data suggested that conserved proteins, which contribute to the capsule-positive phenotype, are potential antivirulence drug targets. Therefore, the results from this study have important biologic and translational implications and, to the best of our knowledge, are the first to address the role of capsule in the pathogenesis of A. baumannii infection

Growth and/or survival of hvKP1 grown in human ascites supplemented with varying concentration of homologous, conditioned ascites.

<p>The growth and/or survival of hvKP1 was assessed at 0, 3, 6, and 24 hours in 100% human ascites supplemented with homologous conditioned ascites resulting in final concentrations of 0%, 1%, 5%, 10%, and 25%. Growth and/or survival was also assessed in ascites supplemented with 25% unconditioned ascites heated at 56°C for 30 minutes to control for a possible effect of conditioned ascites diluting complement-mediated bactericidal activity. <u>Panel A</u>: ascites batch 1. <u>Panel B:</u> ascites batch 2. Supplementation with 5%, 10%, and 25% conditioned, homologous ascites, compared to 0%, resulted in a significant increase in growth and/or survival for both batches of ascites (* P<0.05/4) and supplementation with 1% conditioned, homologous ascites resulted in a trend for increased growth and/or survival (# P>0.05/4 and<0.05). Data are Mean ± S.E.M.; N = 2–3.</p

Neither LuxS generated AI-2 signaling molecules or the AI-3 signaling system appeared to affect the growth and/or survival of hvKP1 in conditioned ascites.

<p>The growth of hvKP1 was assessed at 0, 3, 6, and 24 hours in human ascites (batch 2). <u>Panel A</u>: Ascites was not supplemented (unconditioned) or supplemented with hvKP1 generated conditioned ascites (final concentration 5%) or hvKP1Δ<i>luxS</i> generated conditioned ascites (batch 2) (final concentration 5%). There was no significant difference in the growth of hvKP1 when supplemented with either of these conditioned media. <u>Panel B</u>: Ascites was not supplemented (unconditioned) or supplemented with hvKP1 generated conditioned ascites (batch 2) (final concentration 5%) or epinephrine (final concentrations 50 µM and 500 µM). There was no significant difference in the growth of hvKP1 with the addition of either of these supplements. Data are Mean ± S.E.M.; N = 2–3.</p

Siderophore concentration in hvKP1 and cKP1-4 generated conditioned ascites.

<p>*P<0.05/4 , hvKP1 compared to cKP1-4.</p

The mediators present in hvKP1 generated conditioned minimal medium and ascites that enhance the growth and/or survival of hvKP1 are <3kD and heat stable.

<p>The growth of hvKP1 was assessed at 0, 3, 6, and 24 hours in human ascites (batch 2). <u>Panel A</u>: Ascites was supplemented with either hvKP1 generated conditioned (cond) M9 minimal medium (MM) (10% final concentration) that was: 1) non-fractionated (unfract), 2) <3kD fraction, and 3) >10kD fraction or unconditioned (uncond) minimal medium (10% final concentration) that was: 4) non-fractionated, and 5) <10kD fraction. The growth and/or survival of hvKP1 was significantly increased when the ascites was supplemented with 10% conditioned, non-fractionated minimal medium or with 10% conditioned, <3kD fraction of minimal medium compared to 10% unconditioned, non-fractionated minimal medium and 10% unconditioned, <10kD fraction of minimal medium respectively (* P<0.5/1). Data are Mean ± S.E.M.; N = 2-3. <u>Panel B</u>: Ascites was not supplemented (unconditioned) or supplemented with hvKP1 generated conditioned (cond) ascites or with hvKP1 generated conditioned ascites that was heated at 56°C for 30 minutes (Δ56°C) (batch 2) (5% final concentration for both supplements). The growth and/or survival of hvKP1 was significantly increased when ascites was supplemented with either conditioned or conditioned medium Δ56°C compared to unconditioned ascites (* P<0.05/2). Data are Mean ± S.E.M.; N = 4.</p