SUMMARY
Pathogenic bacteria produce an elaborate assortment of extracellular and cell-associated bacterial products that enable colonization and establishment of infection within a host. Lipopolysaccharide (LPS) molecules are cell surface factors that are typically known for their protective role against serum-mediated lysis and their endotoxic properties. The most heterogeneous portion of LPS is the O antigen or O polysaccharide, and it is this region which confers serum resistance to the organism. Pseudomonas aeruginosa is capable of concomitantly synthesizing two types of LPS referred to as A band and B band. The A-band LPS contains a conserved O polysaccharide region composed of d-rhamnose (homopolymer), while the B-band O-antigen (heteropolymer) structure varies among the 20 O serotypes of P. aeruginosa. The genes coding for the enzymes that direct the synthesis of these two O antigens are organized into two separate clusters situated at different chromosomal locations. In this review, we summarize the organization of these two gene clusters to discuss how A-band and B-band O antigens are synthesized and assembled by dedicated enzymes. Examples of unique proteins required for both A-band and B-band O-antigen synthesis and for the synthesis of both LPS and alginate are discussed. The recent identification of additional genes within the P. aeruginosa genome that are homologous to those in the A-band and B-band gene clusters are intriguing since some are able to influence O-antigen synthesis. These studies demonstrate that P. aeruginosa represents a unique model system, allowing studies of heteropolymeric and homopolymeric O-antigen synthesis, as well as permitting an examination of the interrelationship of the synthesis of LPS molecules and other virulence determinants.
A noninvasive, real-time detection technology was validated for qualitative and quantitative antimicrobial treatment applications. The lux gene cluster of Photorhabdus luminescens was introduced into an Escherichia coli clinical isolate, EC14, on a multicopy plasmid. This bioluminescent reporter bacterium was used to study antimicrobial effects in vitro and in vivo, using the neutropenic-mouse thigh model of infection. Bioluminescence was monitored and measured in vitro and in vivo with an intensified charge-coupled device (ICCD) camera system, and these results were compared to viable-cell determinations made using conventional plate counting methods. Statistical analysis demonstrated that in the presence or absence of antimicrobial agents (ceftazidime, tetracycline, or ciprofloxacin), a strong correlation existed between bioluminescence levels and viable cell counts in vitro and in vivo. Evaluation of antimicrobial agents in vivo could be reliably performed with either method, as each was a sound indicator of therapeutic success. Dose-dependent responses could also be detected in the neutropenic-mouse thigh model by using either bioluminescence or viable-cell counts as a marker. In addition, the ICCD technology was examined for the benefits of repeatedly monitoring the same animal during treatment studies. The ability to repeatedly measure the same animals reduced variability within the treatment experiments and allowed equal or greater confidence in determining treatment efficacy. This technology could reduce the number of animals used during such studies and has applications for the evaluation of test compounds during drug discovery.
Pseudomonas aeruginosa coexpresses two distinct lipopolysaccharide (LPS) molecules known asPseudomonas aeruginosa is an opportunistic pathogen responsible for many debilitating infections, with one of the most notable being chronic pulmonary infections in cystic fibrosis patients. The pathogenicity of this organism is attributed to the production of such diverse virulence factors as exotoxin A, phospholipase C, proteases, alginate, and lipopolysaccharide (LPS). LPS molecules also play an essential structural role in the outer membrane and consist of three distinct regions: a hydrophobic lipid A, which serves to anchor the LPS in the outer membrane, a core oligosaccharide, and the O antigen (O polysaccharide). P. aeruginosa has been shown to coexpress two distinct forms of LPS, known as A band and B band. A-band LPS is an antigenically conserved molecule with an O-polysaccharide region composed of short-chained polymers of D-rhamnose, arranged as trisaccharide repeat units of ␣132, ␣133, and ␣133 linkages (2). In comparison, B-band LPS is serospecific, with variations in the O-antigen structure differentiating P. aeruginosa into 20 distinct serotypes (36,46,47). During chronic pulmonary infections in cystic fibrosis patients, P. aeruginosa isolates become nontypeable due to the loss of B-band O antigen, while A-band LPS and alginate become the primary surface polysaccharides (22, 42). To determine the mechanisms involved in P. aeruginosa LPS biosynthesis and regulation, our laboratory has focused on identifying and characterizing genes involved in A-band and B-band synthesis and expression.
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