One of the hallmarks of Pseudomonas aeruginosa infection in cystic fibrosis (CF) patients is very-high-celldensity (HCD) replication in the lung, allowing this bacterium to induce virulence controlled by the quorumsensing systems. However, the nutrient sources sustaining HCD replication in this chronic infection are largely unknown. Here, we performed microarray studies of P. aeruginosa directly isolated from the lungs of CF patients to demonstrate its metabolic capability and virulence in vivo. In vivo microarray data, confirmed by real-time reverse transcription-PCR, indicated that the P. aeruginosa population expressed several genes for virulence, drug resistance, and utilization of multiple nutrient sources (lung surfactant lipids and amino acids) contributing to HCD replication. The most abundant lung surfactant lipid molecule, phosphatidylcholine (PC), induces key genes of P. aeruginosa pertinent to PC degradation in vitro as well as in vivo within the lungs of CF patients. The results support recent research indicating that P. aeruginosa exists in the lungs of CF patients as a diverse population with full virulence potential. The data also indicate that there is deregulation of several pathways, suggesting that there is in vivo evolution by deregulation of a large portion of the transcriptome during chronic infection in CF patients. To our knowledge, this is the first in vivo transcriptome analysis of P. aeruginosa in a natural infection in CF patients, and the results indicate several important aspects of P. aeruginosa pathogenesis, drug resistance, nutrient utilization, and general metabolism within the lungs of CF patients.Pseudomonas aeruginosa is the major cause of morbidity and mortality in lung diseases, including cystic fibrosis (CF) (6, 11, 32) and nosocomial pneumonia (3,40). Over 93% of CF patients between 18 and 24 years old have been reported to have P. aeruginosa infections (11). In addition, nosocomial pneumonia is the second most common of all nosocomial infections, and P. aeruginosa was the most frequently isolated microbe involved from 1992 to 1997 (37). The pathogenesis of this organism has been intensively studied with respect to virulence and virulence expression (5,26,31,36,39), biofilm production (10, 43), and quorum sensing (15,27,28,30). Several virulence factors that P. aeruginosa expresses (e.g., exotoxin A, exoenzyme S, cytotoxin, proteases, lipases, phospholipases, alginate, and hydrogen cyanide) all contribute to severe lung damage. High-cell-density (HCD) replication is necessary for many of these events to occur, and Ͼ10 9 bacteria/ml of sputum have been found in the lungs of CF patients (43, 46). The ability of P. aeruginosa to obtain nutrients in the lung for HCD replication and maintenance is the quintessential factor leading to quorum-sensing-induced virulence expression, which is a hallmark of chronic lung infections in CF patients. However, the nutrient requirements of P. aeruginosa in vivo are unknown. A recent in vitro study by Palmer et al. (25), in which P. aerugi...

Vibrio cholerae serogroup O1, the causative agent of the diarrheal disease cholera, is divided into two biotypes: classical and El Tor. Both biotypes produce the major virulence factors toxin-coregulated pilus (TCP) and cholera toxin (CT). Although possessing genotypic and phenotypic differences, El Tor biotype strains displaying classical biotype traits have been reported and subsequently were dubbed El Tor variants. Of particular interest are reports of El Tor variants that produce various levels of CT, including levels typical of classical biotype strains. Here, we report the characterization of 10 clinical isolates from the International Centre for Diarrhoeal Disease Research, Bangladesh, and a representative strain from the 2010 Haiti cholera outbreak. We observed that all 11 strains produced increased CT (2-to 10-fold) compared to that of wild-type El Tor strains under in vitro inducing conditions, but they possessed various TcpA and ToxT expression profiles. Particularly, El Tor variant MQ1795, which produced the highest level of CT and very high levels of TcpA and ToxT, demonstrated hypervirulence compared to the virulence of El Tor wild-type strains in the infant mouse cholera model. Additional genotypic and phenotypic tests were conducted to characterize the variants, including an assessment of biotype-distinguishing characteristics. Notably, the sequencing of ctxB in some El Tor variants revealed two copies of classical ctxB, one per chromosome, contrary to previous reports that located ctxAB only on the large chromosome of El Tor biotype strains.Vibrio cholerae is a Gram-negative, curved-rod-shaped bacterium that is the causative agent of the watery diarrheal disease cholera. The structure of the cell surface lipopolysaccharide O antigen is used to classify V. cholerae into more than 200 serogroups, of which only two, O1 and O139, possess the potential to cause epidemic or pandemic cholera. The O1 serogroup is further divided into two biotypes, classical and El Tor, which evolved from independent lineages (20, 22), and they display genotypic and phenotypic differences.V. cholerae O1 is distinguished by two of its major virulence factors, cholera toxin (CT) and the toxin-coregulated pilus (TCP). The cholera toxin is encoded by ctxA and ctxB, which are found on the CTX prophage (49), and is responsible for the manifestation of diarrheal disease with severe water and electrolyte loss. The TCP, encoded by the tcp operon in the Vibrio pathogenicity island (VPI), is required for V.

Allelic replacement in the Burkholderia genus has been problematic due to the lack of appropriate counterselectable and selectable markers. The counter-selectable marker sacB, commonly used in gram-negative bacteria, is nonselective on sucrose in many Burkholderia species. In addition, the use of antibiotic resistance markers of clinical importance for the selection of desirable genetic traits is prohibited in the United States for two potential bioterrorism agents, Burkholderia mallei and Burkholderia pseudomallei. Here, we engineered a mutated counter-selectable marker based on the B. pseudomallei PheS (the ␣-subunit of phenylalanyl tRNA synthase) protein and tested its effectiveness in three different Burkholderia species. The mutant PheS protein effectively killed 100% of the bacteria in the presence of 0.1% p-chlorophenylalanine. We assembled the mutant pheS on several allelic replacement vectors, in addition to constructing selectable markers based on tellurite

The Pseudomonas aeruginosa PsrA responds to long-chain fatty acid signals to regulate the fadBA5 b-oxidation operon b-Oxidative enzymes for fatty acid degradation (Fad) of long-chain fatty acids (LCFAs) are induced in vivo during lung infection in cystic fibrosis patients, and this may contribute to nutrient acquisition and pathogenesis of Pseudomonas aeruginosa. The promoter region of one P. aeruginosa b-oxidation operon, fadBA5 (PA3014 and PA3013), was mapped. Focusing on the transposon mutagenesis of strain PAO1 carrying the P fadBA5 -lacZ fusion, a regulator for the fadBA5 operon was identified to be PsrA (PA3006). Transcriptome analysis of the DpsrA mutant indicated its importance in regulating b-oxidative enzymes. These microarray data were confirmed by real-time RT-PCR analyses of the fadB5 and lipA (encoding a lipase) genes. Induction of the fadBA5 operon was demonstrated to respond to novel LCFA signals, and this induction required the presence of PsrA, suggesting that LCFAs bind to PsrA to derepress fadBA5. Electrophoretic mobility shift assays indicate specific binding of PsrA to the fadBA5 promoter region. This binding is disrupted by specific LCFAs (C 18 : 1 D9 , C 16 : 0 , C 14 : 0 and, to a lesser extent, C 12 : 0 ), but not by other medium-or short-chain fatty acids or the first intermediate of b-oxidation, acyl-CoA. It is shown here that PsrA is a fadBA5 regulator that binds and responds to LCFA signals in P. aeruginosa. INTRODUCTIONPseudomonas aeruginosa is an opportunistic pathogen, and the spectrum of infections and diseases it causes is second to none. P. aeruginosa can cause infections of the ear, bone, joint, skin and soft tissue, and more serious infections, including meningitis, bacteraemia, endocarditis, ocular infections, hospital-acquired pneumonia, and lung infections in cystic fibrosis (CF) patients (Baltch & Griffin, 1977;Bowton, 1999;Fleiszig et al., 1995;Greenberger, 1997;Lode et al., 2000;Pruitt et al., 1998;Reyes & Lerner, 1983;Richards et al., 1999;Schaberg et al., 1991). P. aeruginosa lung infections transpire in nosocomial pneumonia and in CF patients. Nosocomial pneumonia is the second most common of all nosocomial infections, and P. aeruginosa has been the most frequently isolated microbe responsible (Pennington, 1994;Richards et al., 1999). In addition, over 93 % of CF patients between the ages of 18 and 24 have been infected with P. aeruginosa (Doring, 1997).Fatty acid degradation (Fad) pathways may play critical roles in P. aeruginosa pathogenesis. It has recently been demonstrated that the Fad pathways of P. aeruginosa may have important implications in nutrient acquisition and pathogenesis within the CF lung (Son et al., 2007), through the degradation of an essential lung surfactant component phosphatidylcholine (PC). The degradation of one PC component, long-chain fatty acid (LCFA), potentially occurs through three different b-oxidation pathways, involving three fadBA operons (fadAB1, PA1737 and PA1736; fadAB4, PA4786 and PA4785; and fadBA5, PA3014 and PA3013) (Son e...

Kilham rat virus (KRV) causes autoimmune diabetes in diabetes-resistant BioBreeding (DR-BB) rats; however, the mechanism by which KRV induces autoimmune diabetes without the direct infection of β cells is not well understood. We first asked whether molecular mimicry, such as a common epitope between a KRV-specific peptide and a β cell autoantigen, is involved in the initiation of KRV-induced autoimmune diabetes in DR-BB rats. We found that KRV peptide-specific T cells generated in DR-BB rats infected with recombinant vaccinia virus expressing KRV-specific structural and nonstructural proteins could not induce diabetes, indicating that molecular mimicry is not the mechanism by which KRV induces autoimmune diabetes. Alternatively, we asked whether KRV infection of DR-BB rats could disrupt the finely tuned immune balance and activate autoreactive T cells that are cytotoxic to β cells, resulting in T cell-mediated autoimmune diabetes. We found that both Th1-like CD45RC+CD4+ and cytotoxic CD8+ T cells were up-regulated, whereas Th2-like CD45RC−CD4+ T cells were down-regulated, and that isolated and activated CD45RC+CD4+ and CD8+ T cells from KRV-infected DR-BB rats induced autoimmune diabetes in young diabetes-prone BioBreeding (DP-BB) rats. We conclude that KRV-induced autoimmune diabetes in DR-BB rats is not due to molecular mimicry, but is due to a breakdown of the finely tuned immune balance of Th1-like CD45RC+CD4+ and Th2-like CD45RC−CD4+ T cells, resulting in the selective activation of β cell-cytotoxic effector T cells.