Nicole R. Luke

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

ABSTRACT 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.

Identification and Characterization of an Acinetobacter baumannii Biofilm-Associated Protein

We have identified a homologue to the staphylococcal biofilm-associated protein (Bap) in a bloodstream isolate of Acinetobacter baumannii. The fully sequenced open reading frame is 25,863 bp and encodes a protein with a predicted molecular mass of 854 kDa. Analysis of the nucleotide sequence reveals a repetitive structure consistent with bacterial cell surface adhesins. Bap-specific monoclonal antibody (MAb) 6E3 was generated to an epitope conserved among 41% of A. baumannii strains isolated during a recent outbreak in the U.S. military health care system. Flow cytometry confirms that the MAb 6E3 epitope is surface exposed. Random transposon mutagenesis was used to generate A. baumannii bap1302::EZ-Tn5, a mutant negative for surface reactivity to MAb 6E3 in which the transposon disrupts the coding sequence of bap. Time course confocal laser scanning microscopy and three-dimensional image analysis of actively growing biofilms demonstrates that this mutant is unable to sustain biofilm thickness and volume, suggesting a role for Bap in supporting the development of the mature biofilm structure. This is the first identification of a specific cell surface protein directly involved in biofilm formation by A. baumannii and suggests that Bap is involved in intercellular adhesion within the mature biofilm.

Contribution of Moraxella catarrhalis Type IV Pili to Nasopharyngeal Colonization and Biofilm Formation

ABSTRACT Moraxella catarrhalis is a gram-negative mucosal pathogen of the human respiratory tract. Although little information is available regarding the initial steps of M. catarrhalis pathogenesis, this organism must be able to colonize the human mucosal surface in order to initiate an infection. Type IV pili (TFP), filamentous surface appendages primarily comprised of a single protein subunit termed pilin, play a crucial role in the initiation of disease by a wide range of bacteria. We previously identified the genes that encode the major proteins involved in the biosynthesis of M. catarrhalis TFP and determined that the TFP expressed by this organism are highly conserved and essential for natural transformation. We extended this initial study by investigating the contribution of TFP to the early stages of M. catarrhalis colonization. TFP-deficient M. catarrhalis bacteria exhibit diminished adherence to eukaryotic cells in vitro. Additionally, our studies demonstrate that M. catarrhalis cells form a mature biofilm in continuous-flow chambers and that biofilm formation is enhanced by TFP expression. The potential role of TFP in colonization by M. catarrhalis was further investigated using in vivo studies comparing the abilities of wild-type M. catarrhalis and an isogenic TFP mutant to colonize the nasopharynx of the chinchilla. These results suggest that the expression of TFP contributes to mucosal airway colonization. Furthermore, these data indicate that the chinchilla model of nasopharyngeal colonization provides an effective animal system for studying the early steps of M. catarrhalis pathogenesis.

Expression of Type IV Pili by Moraxella catarrhalis Is Essential for Natural Competence and Is Affected by Iron Limitation

ABSTRACT Type IV pili, filamentous surface appendages primarily composed of a single protein subunit termed pilin, play a crucial role in the initiation of disease by a wide range of pathogenic bacteria. Although previous electron microscopic studies suggested that pili might be present on the surface of Moraxella catarrhalis isolates, detailed molecular and phenotypic analyses of these structures have not been reported to date. We identified and cloned the M. catarrhalis genes encoding PilA, the major pilin subunit, PilQ, the outer membrane secretin through which the pilus filament is extruded, and PilT, the NTPase that mediates pilin disassembly and retraction. To initiate investigation of the role of this surface organelle in pathogenesis, isogenic pilA, pilT, and pilQ mutants were constructed in M. catarrhalis strain 7169. Comparative analyses of the wild-type 7169 strain and three isogenic pil mutants demonstrated that M. catarrhalis expresses type IV pili that are essential for natural genetic transformation. Our studies suggest type IV pilus production by M. catarrhalis is constitutive and ubiquitous, although pilin expression was demonstrated to be iron responsive and Fur regulated. These data indicate that additional studies aimed at elucidating the prevalence and role of type IV pili in the pathogenesis and host response to M. catarrhalis infections are warranted.

Identification and Characterization of a Glycosyltransferase Involved in Acinetobacter baumannii Lipopolysaccharide Core Biosynthesis

ABSTRACT Although Acinetobacter baumannii has emerged as a significant cause of nosocomial infections worldwide, there have been few investigations describing the factors important for A. baumannii persistence and pathogenesis. This paper describes the first reported identification of a glycosyltransferase, LpsB, involved in lipopolysaccharide (LPS) biosynthesis in A. baumannii. Mutational, structural, and complementation analyses indicated that LpsB is a core oligosaccharide glycosyl transferase. Using a genetic approach, lpsB was compared with the lpsB homologues of several A. baumannii strains. These analyses indicated that LpsB is highly conserved among A. baumannii isolates. Furthermore, we developed a monoclonal antibody, monoclonal antibody 13C11, which reacts to an LPS core epitope expressed by approximately one-third of the A. baumannii clinical isolates evaluated to date. Previous studies describing the heterogeneity of A. baumannii LPS were limited primarily to structural analyses; therefore, studies evaluating the correlation between these surface glycolipids and pathogenesis were warranted. Our data from an evaluation of LpsB mutant 307::TN17, which expresses a deeply truncated LPS glycoform consisting of only two 3-deoxy-d-manno-octulosonic acid residues and lipid A, suggest that A. baumannii LPS is important for resistance to normal human serum and confers a competitive advantage for survival in vivo. These results have important implications for the role of LPS in A. baumannii infections.

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

ABSTRACT 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.

Identification of a 3-Deoxy-d-manno-Octulosonic Acid Biosynthetic Operon in Moraxella catarrhalis and Analysis of a KdsA-Deficient Isogenic Mutant

ABSTRACT Lipooligosaccharide (LOS), a predominant surface-exposed component of the outer membrane, has been implicated as a virulence factor in the pathogenesis of Moraxella catarrhalis infections. However, the critical steps involved in the biosynthesis and assembly of M. catarrhalis LOS currently remain undefined. In this study, we used random transposon mutagenesis to identify a 3-deoxy-d-manno-octulosonic acid (KDO) biosynthetic operon in M. catarrhalis with the gene order pyrG-kdsA-eno. The lipid A-KDO molecule serves as the acceptor onto which a variety of glycosyl transferases sequentially add the core and branch oligosaccharide extensions for the LOS molecule. KdsA, the KDO-8-phosphate synthase, catalyzes the first step of KDO biosynthesis and is an essential enzyme in gram-negative enteric bacteria for maintenance of bacterial viability. We report the construction of an isogenic M. catarrhalis kdsA mutant in strain 7169 by allelic exchange. Our data indicate that an LOS molecule consisting only of lipid A and lacking KDO glycosylation is sufficient to sustain M. catarrhalis survival in vitro. In addition, comparative growth and susceptibility assays were performed to assess the sensitivity of 7169kdsA11 compared to that of the parental strain. The results of these studies demonstrate that the native LOS molecule is an important factor in maintaining the integrity of the outer membrane and suggest that LOS is a critical component involved in the ability of M. catarrhalis to resist the bactericidal activity of human sera.

Identification of a Novel Two-Partner Secretion Locus in Moraxella catarrhalis

ABSTRACT Although Moraxella catarrhalis continues to be a significant cause of disease in both children and adults, the steps involved in pathogenesis remain poorly understood. We have identified three open reading frames in the M. catarrhalis genome that encode homologues of the two-partner secretion system (TPS). The sequenced M. catarrhalis hemagglutinin-like locus of strain 7169 has a unique gene organization composed in the order of mchA1, mchB, and mchA2, where mchA1 is divergent. MchA1 and MchA2 are 74% identical at the amino acid level and diverge only in the C-terminal regions. The TPS motif identified in the common N-terminal regions of MchA1 and MchA2 was found to be homologous to the filamentous hemagglutinin of Bordetella pertussis, and MchB has homology to other TpsB transporters. The presence of MchA1 and MchA2 in outer membrane protein preparations and concentrated culture supernatants (CCSs) of strain 7169 was confirmed by immunoblotting using specific antisera. Nanoscale liquid chromatography-tandem mass spectrometry peptide sequencing of the antibody-reactive bands from the CCSs was performed and demonstrated that 13 different peptides mapped to identical regions of MchA1 and MchA2. Quantitative adherence assays revealed a decrease of binding to primary normal human bronchial epithelial cells by the mch mutants 7169mchB and 7169mchA1A2B compared to that by the wild-type strain. These studies show that MchA1, MchA2, and MchB are components of a novel TPS identified in M. catarrhalis and suggest that these proteins may be involved in colonization.

Inactivation of the Moraxella catarrhalis Superoxide Dismutase SodA Induces Constitutive Expression of Iron-Repressible Outer Membrane Proteins

ABSTRACT Many pathogens produce one or more superoxide dismutases (SODs), enzymes involved in the detoxification of endogenous and exogenous reactive oxygen species that are encountered during the infection process. One detectable cytoplasmic SOD was identified in the human mucosal pathogen Moraxella catarrhalis, and the gene responsible for the SOD activity, sodA, was isolated from a recent pediatric clinical isolate (strain 7169). Sequence analysis of the cloned M. catarrhalis 7169 DNA fragment revealed an open reading frame of 618 bp encoding a polypeptide of 205 amino acids with 48 to 67% identity to known bacterial manganese-cofactored SODs. An isogenic M. catarrhalis sodA mutant was constructed in strain 7169 by allelic exchange. In contrast to the wild-type 7169, the 7169::sodK20 mutant was severely attenuated for aerobic growth, even in rich medium containing supplemental amino acids, and exhibited extreme sensitivity to the redox-active agent methyl viologen. The ability of recombinant SodA to rescue the aerobic growth defects of E. coli QC774, a sodA sodB-deficient mutant, demonstrated the functional expression of SOD activity by cloned M. catarrhalis sodA. Indirect SOD detection assays were used to visualize both native and recombinant SodA activity in bacterial lysates. This study demonstrates that M. catarrhalis SodA plays a critical role in the detoxification of endogenous, metabolically produced oxygen radicals. In addition, the outer membrane protein (OMP) profile of 7169::sodK20 was consistent with iron starvation in spite of growth under iron-replete conditions. This novel observation indicates that M. catarrhalis strains lacking SodA constitutively express immunogenic OMPs previously described as iron repressible, and this potentially attenuated mutant strain may be an attractive vaccine candidate.