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.

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.

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.

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.Moraxella catarrhalis, a gram-negative mucosal pathogen of the human respiratory tract, can also reside as a nasopharyngeal commensal. M. catarrhalis is capable of causing multiple diseases of the upper respiratory tract, including sinusitis, laryngitis, and acute and chronic otitis media, predominantly in pediatric populations, as well as recurrent exacerbations of chronic obstructive pulmonary disease in adults with underlying lung disease (31, 41). As with many other pathogenic bacteria, adherence to host tissues and subsequent colonization of the respiratory tract mucosa are believed to be essential prior to the development of M. catarrhalis infections.M. catarrhalis expresses a number of cell surface components that have been postulated to be involved in bacterial virulence (reviewed in references 29 and 41). However, in vivo studies of M. catarrhalis pathogenesis have been limited by the lack of an effective animal model to investigate bacterial colonization of the upper respiratory tract epithelium. Even with the most prevalently used model system, the mouse pulmonary clearance model, M. catarrhalis is markedly cleared from the site within 6 h and the organism is almost completely eradicated by 24 h (40). Although little information is available regarding the actual steps involved in the pathogenesis of M. catarrhalis infections in vivo, it is clear that this organism must attach to the mucosal surface in order to ...

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.

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.Moraxella catarrhalis is now recognized as an important human pathogen in both children and adults (12,16,19,33,49). This organism is a significant cause of otitis media and sinusitis in young children and also causes lower respiratory tract disease in adults, particularly those with chronic lung disease (12,33,34). In addition, a few reports have described nosocomial spread of this bacterium in respiratory wards (6-9, 35). The extremely high carriage rates reported in children, coupled with the fact that over 90% of M. catarrhalis clinical isolates are ␤-lactamase positive, suggest that infections with these organisms may increase (12,48). Multiple studies, including those from our laboratory, have described specific bacterial components as potential virulence factors (for a recent review, refer to reference 49). Although little information is available regarding the actual steps involved in the pathogenesis of M. catarrhalis infections, it is clear that these organisms must attach to the human mucosal surface in order to establish colonization. Therefore, the identification of bacterial colonization factors and of new vaccine and treatment targets is a major focus of present research efforts. In this study, we describe the identification and characterization of the genes that are involved in the biosynthesis and assembly of M. catarrhalis pili.Pili are homo-or heteropolymers composed of helically arranged subunits assembled and expressed on the surface of a broad spectrum of gram-negative bacteria and can be classified based on morphology and function.

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.Moraxella catarrhalis is a gram-negative aerobic diplococcus that is frequently identified as part of the nasopharyngeal floras, particularly in pediatric populations (4). This bacterium is an important mucosal pathogen of the upper and lower respiratory tracts in humans. In particular, the organism is a leading cause of otitis media and sinusitis in young children and is associated with pulmonary exacerbations in adults with chronic lung disease or compromised immune function (26, 27). Research over the past decade has focused on the identification and characterization of M. catarrhalis surface antigens, including lipooligosaccharides (LOS), as potential vaccine candidates (for recent reviews, see references 17, 22, 23, and 42).LOS, a predominant surface-exposed component of the outer membrane, has been implicated as a virulence factor in the pathogenesis of M. catarrhalis infections. The LOS of M. catarrhalis is similar to that of other mucosal pathogens in that it lacks a repeating O-antigen attached to the core oligosaccharide, which is characteristic of the lipopolysaccharide (LPS) molecule. Instead, the LOS molecule contains a lipid A-proximal conserved inner core and one or more structurally diverse oligosaccharide branch extensions that determine serologic specificity. Although the LOS of M. catarrhalis appears to be more antigenically conserved than the LOS of other bacteria, three LOS serotypes (termed A, B, and C) have been identified on the basis of structural and immunologic analyses ...

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) Moraxella catarrhalis is an important gram-negative human mucosal pathogen. It is one of the three major causes of acute otitis media, along with Streptococcus pneumoniae and nontypeable Haemophilus influenzae (10, 43). In adults with chronic bronchitis and chronic obstructive pulmonary disease (COPD), M. catarrhalis causes lower respiratory tract infections that often lead to acute exacerbations (33, 34). In addition, M. catarrhalis can cause sinusitis in infants and young children (23,43). Because 90% of M. catarrhalis clinical isolates are beta-lactamase positive (23,43) and no protective vaccine is available (29, 30), M. catarrhalis continues to be a major source of human disease..Most of the research involving M. catarrhalis pathogenesis has focused largely on the identification and characterization of outer membrane proteins (OMPs) on the bacterial surface, although most of these have an undefined role in virulence (23,32,43). M. catarrhalis expresses some OMPs, including the transferrin binding protein TbpB (28) and the hemin and hemoglobin utilization proteins HumA and MhuA (11,12), to obtain iron from the human host. In addition, other OMPs, such as the ubiquitous surface protein UspA2, can be involved in serum resistance (1) or in natural competence, as described for the type IV pilus (26). To date, only a few OMPs, including UspA1 and UspA2H (1, 24), the M. catarrhalis adherence protein McaP (41), and the hemagglutinating protein Hag/M. catarrhalis immunoglobulin D-binding protein (3,14,19), have been reported to directly mediate binding to cell lines in vitro. Therefore, it is clear that like many other gram-negative pathogens, M. catarrhalis has developed multiple virulence mechanisms to successfully colonize the human mucosal surface.In this report, we have identified a locus in M. catarrhalis 7169 containing three open reading frames (ORFs) that encode homologues of the previously described two-partner secretion systems (TPS) in various other pathogens, including Bordetella pertussis (21,22). The B. pertussis TPS pathway is composed of the filamentous hemagglutinin FhaB (generically named TpsA) and the transporter FhaC (TpsB) (25). FhaB is the major adhesin involved in bacterial attachment and colonization of the human upper respiratory tract, and this protein is also a component of the acellular diphtheria-pertussis vaccine (25, 36). The M. catarrhalis hemagglutinin-like locus described in this study contains three ORFs, termed mchA1, mchA2, and mchB. The TPS motif identified in MchA1 and MchA2 was found to be homologous to FhaB of B. pertussis. MchB has homology to FhaC of B. pertussis (22). This is the first report of a TPS in M. catarrhalis, and our data demonstrate that this system is likely...