Campus Box 7248
Chapel Hill, NC 27599-7248
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen responsible for a variety of diseases in individuals with compromised immune function. This organism poses the greatest risk to the hospitalized population, the elderly, immunosuppressed individuals and those with co-morbid illness, such as heart and pulmonary disease, diabetes, cancer and AIDS. The medical importance of this organism is further underscored by the fact that it is the primary cause of morbidity and mortality in individuals with cystic fibrosis (CF) where it causes chronic lung infection. The ability of P. aeruginosa to cause this wide variety of diseases depends on the expression of an array of virulence factors that are associated with the bacterial surface or are secreted into the local environment in response to specific host cues. Further, it appears that differential progression of acute or chronic P. aeruginosa infections involves production of distinct sets of virulence factors. Research in our laboratory focuses on the molecular mechanisms employed by P. aeruginosa to cause different diseases, with emphasis on the signal transduction pathways that regulate virulence factor expression in response to the host environment.
Coordinated Regulation of P. aeruginosa Virulence
We have shown that synthesis of the second-messenger signaling molecule adenosine 3’, 5’-cyclic monophosphate (cyclic AMP or cAMP) is required for P. aeruginosa virulence. Mutants lacking CyaB, an adenylate cyclase, produce significantly reduced levels of cAMP, are not cytotoxic to cultured epithelial cells, do not infect well-differentiated human airway epithelial cultures and are highly attenuated in an adult mouse model of acute pneumonia.
In P. aeruginosa, cAMP generates a transcriptional response by binding to and activating the transcription factor Vfr (Virulence factor regulator). Microarray analysis indicates that the cAMP-Vfr complex controls the expression of nearly 200 P. aeruginosa genes, the majority of which encode virulence factors required for host colonization, surface motility, biofilm formation and the secretion and delivery of toxins (Figure 1). We propose that the cAMP/Vfr signaling pathway plays a central role in detecting the host environment and in initiating a coordinated pathogenic response.
Figure 1. The P. aeruginosa cAMP-signaling cascade coordinates the expression of virulence genes. The membrane bound adenylate cyclase CyaB is regulated by environmental cues. Its product, cAMP, acts as a cofactor for Vfr, which coordinates the expression of genes encoding components of key bacterial virulence determinants.
Projects / Future goals
Reciprocal regulation of acute and chronic virulence factors. P. aeruginosa isolated from patients with acute respiratory infection are generally non-encapsulated and express a variety of invasive virulence factors including flagella, the type III secretion system, type IV pili, and multiple secreted toxins and degradative enzymes. Strains isolated from chronically infected CF patients, however, typically lack expression of invasive virulence factors and have a mucoid phenotype due to production of an alginate capsule. The mucoid phenotype primarily results from loss-of-function mutations in MucA, an anti-sigma factor that normally prevents alginate synthesis. We discovered that mucA mutants do not express vfr, demonstrating that a single naturally occurring mutation leads to inverse regulation of virulence factors involved in the acute and chronic phases of CF airway infection. Further, these results suggest that in the context of the CF lung, mucoid conversion and inhibition of invasive virulence determinants may both confer a selective advantage to mucA mutant strains of P. aeruginosa.
Regulation of cAMP homeostasis. In order for cAMP to be an effective second-messenger signaling molecule its intracellular levels must be tightly regulated. Three general mechanisms control intracellular levels of cAMP in bacteria: synthesis, degradation and transport (Figure 2). We are interested in understanding how each of these processes are regulated and the role they play in controlling P. aeruginosa virulence gene expression.
Figure 2. Cyclic AMP control mechanisms. Intracelluar cAMP levels are controlled at the level of synthesis (adenylate cyclase), degradation (cAMP phosphodiesterase) and transport. P. aeruginosa exhibits all three control mechanisms. Cyclic AMP is produced by two adenylate cyclases, CyaA and CyaB, with CyaB responsible for the majority of cAMP production. Cyclic AMP is degraded by a cAMP phosphodiesterase termed CpdA and genes affecting transport have been identified but the mechanism and relative contribution are unknown.
Regulated Synthesis of cAMP. In P. aeruginosa, cAMP is primarily synthesized by CyaB, a membrane-associated adenylate cyclase. To minimize response time in a rapidly changing environment, adenylate cyclase enzymes are primarily regulated at the functional level. We discovered that CyaB activity is controlled by a chemotaxis-like chemosensing (Chp) signal transduction system that was previously implicated in regulating type IV pili (Figure 3). This finding defines a novel function for a chemotaxis-like system in controlling cAMP production and establishes a regulatory link between the Chp system and cAMP-dependent virulence systems. We are continuing to delineate the molecular signaling events that lead to cAMP synthesis and ultimately activation of the P. aeruginosa virulence program. In addition, we are carrying out a structure/function analysis of CyaB to determine how enzymatic activity is controlled by specific environmental signals.
Figure 3. The Chp chemotaxis-like chemosensing system positively and negatively regulates intracellular cAMP levels. The P. aeruginosa Chp proteins are predicted to be homologs of the E. coli Che system proteins controlling flagellar motility. The corresponding E. coli homolog for each Chp protein is indicated in parentheses: i) PilJ, a member of the transmembrane methyl-accepting chemotaxis protein (MCP) receptor family, ii) ChpA, a complex signal transduction protein with predicted histidine kinase activity (CheA), multiple phosphotransfer domains, a CheW-like domain, and a CheY-like receiver domain, iii) PilI and ChpC, CheW-like accessory proteins, iv) PilG and PilH, CheY-like response regulators, v) PilK, a methyltransferase, and vi) ChpB, a methylesterase. The predicted flow of information in the Chp system is from the PilJ receptor to ChpA (a process mediated by PilI and/or ChpC) and then from ChpA to PilG and PilH. The phosphorylated response regulators are predicted to impart their regulatory effect on the machinery responsible for motility; however, the mechanical basis for type IV pilus-dependent motility in P. aeruginosa is not understood. Methylation of the MCP by PilK promotes signal transduction while ChpB is involved in signal attenuation by mediating MCP demethylation. Our preliminary results suggest that PilH, ChpB and PilK are inhibitors of the cAMP/Vfr signaling pathway while all other components are activators. Figure adapted from Fulcher et al. 2010.
cAMP degradation and transport. Because cAMP is a stable, membrane impermeable molecule signal attenuation is necessary to reset the signaling system and avoid a perpetual response. This function is typically achieved through degradation of cAMP and/or excretion by an unidentified active transport mechanism. We have identified a gene (cpdA) in P. aeruginosa encoding a cAMP phosphodiesterase that mediates cAMP degradation. Deletion of cpdA results in accumulation of intracellular cAMP and altered regulation of P. aeruginosa virulence traits. In addition, we have identified numerous genes whose products significantly influence cAMP transport. Our current effort is to understand how genes affecting cAMP degradation and transport affect P. aeruginosa virulence in vivo.
Vfr-dependent virulence gene regulation. Although indirect evidence suggests that Vfr activity is controlled by cAMP, it has been hypothesized that the cAMP-binding pocket of Vfr may accommodate additional cyclic nucleotides. We recently showed that cAMP was the only cyclic nucleotide capable of restoring DNA binding activity to apo-Vfr. While cAMP is required for Vfr-dependent regulation of multiple virulence genes, we identified one system that was cAMP-independent. The existence of both cAMP-dependent and -independent modes of Vfr regulation suggests that P. aeruginosa can fine-tune its Vfr-dependent virulence program in response to specific host cues or environments. We also defined a role for Vfr in autoregulation of its own (vfr) promoter and in regulation of the cAMP phosphodiesterase gene cpdA, demonstrating that genes involved in cAMP homeostasis and virulence factor expression are subject to regulatory feedback in response to fluctuations in intracellular cAMP levels.
Type IV pilus biogenesis, structure and function. Type IV pili are dynamic, multi-functional bacterial surface fibers responsible for mediating P. aeruginosa interaction with host cells. The process of pilus biogenesis and subsequent extension and retraction of the fiber requires more than 40 genes. We have shown that type IV pili are regulated by cAMP/Vfr at the level of pilus biogenesis gene expression. In addition, our investigation of cAMP regulation by the Chp chemotaxis-like chemosensory system revealed that the Chp system controls TFP production through modulation of cAMP.
Several of our studies have focused on PilY1, which is encoded by the fimUpilVWXY1Y2E pilus biogenesis operon. PilY1 was previously shown to be associated with the pilus fiber and we discovered that it plays a critical role in pilus extension and retraction. Structural analysis of PilY1 revealed that it is a calcium binding protein (Figure 4) and that calcium binding and release control pilus fiber extension and retraction, respectively. We also showed that PilY1 has an additional role in promoting type IV pilus-dependent bacterial adhesion to host tissue. These studies, which were conducted in a mucosal epithelium model system derived from primary human tissue, also revealed that invasion and fulminant infection of intact host tissue requires the coordinated and mutually dependent action of multiple bacterial factors, including pilus retraction and type III secretion (Figure 5).
Figure 4. Strucuture of the C-terminal domain of P. aeruginosa PilY1. The PilY1 structure reveals a modified beta-propeller fold (A) and novel EF-hand-like calcium-binding domain (B). Figure adapted from Orans J et al. 2010.
Figure 5. Model of events leading to invasive infection of the human airway epithelium by P. aeruginosa. (A) Histological cross-sections of HAE cultures prior to infection of following inoculated with wild type P. aeruginosa. After breaching the mucosal barrier, P. aeruginosa interacted efficiently with the basolateral surfaces of ciliated cells and eventually spreads between cells to encompass the entire mucosal epithelium. (B) The three different phases of epithelial infection (apical interaction, penetration and dissemination) and the P. aeruginosa factors required for each phase are indicated. Apical interaction. Productive interactions between P. aeruginosa and the intact mucosal epithelium is a relatively rare event exploiting transiently exposed basolateral surfaces during cell extrusion. This event is dependent on bacterial adherence mediated by TFP and TFP-associated PilY1. Penetration. Subsequent penetration of the mucosal barrier requires retractile TFP and T3S. PilT-dependent pilus retraction may facilitate contact-dependent T3S and TFP-mediated bacterial motility. Cytotoxicity mediated by T3S causes additional tissue damage and disruption of host cell junctions and exposure of additional basolateral host receptors. Dissemination. Following the formation of a focal infection, PilY1-mediated adherence, retractile TFP and T3S act synergistically to cause fulminant infection and dissemination into deeper tissue. Figure adapted from Heiniger et al. 2010.
Tunney MM, Klem ER, Fodor AA, Gilpin DF, Moriarty TF, McGrath SJ, Muhlebach MS, Boucher RC, Cardwell C, Doering G, Elborn JS and Wolfgang MC. (2011) Use of culture and molecular analysis to determine the effect of antibiotic treatment on microbial community diversity and abundance during exacerbation in cystic fibrosis patients. Thorax. 26 Jan 2011. [Epub ahead of print]
Stressmann FA, Rogers GB, Klem ER, Lilley AK, Donaldson SH, Daniels TW, Carroll MP, Patel N, Forbes B, Boucher RC, Wolfgang MC and Bruce KD. (2011) Analysis of the bacterial communities present in the lungs of Cystic Fibrosis patients from American and British centers. J. Clin Microbiol. 49(1): 281-291.
Jones AK, Fulcher NB, Balzer GJ, Urbanowski ML, Pritchett CL, Schurr MJ, Yahr TL and Wolfgang MC. (2010) Activation of the Pseudomonas aeruginosa AlgU regulon through mucA mutation inhibits cAMP/Vfr signaling. J Bacteriol. 192(21): 5709-5717.
Su H-C, Ramkissoon, K, Doolittle J, Clark M, Khatun J, Secrest A, Wolfgang MC and Giddings MC. (2010) The development of ciprofloxacin resistance in Pseudomonas aeruginosa involves multiple response stages and multiple proteins. Antimicrob Agents Chemother. 54(11): 4626-4635.
Heiniger RW, Winther-Larsen HC, Pickles RJ, Koomey M and Wolfgang MC. (2010) Infection of human mucosal tissue by Pseudomonas aeruginosa requires sequential and mutually dependent virulence factors and a novel pilus-associated adhesin. Cell Microbiol. 12(8): 1158-1173.
Fuchs EL, Brutinel ED, Jones AK, Fulcher NB, Urbanowski ML, Yahr TL and Wolfgang MC. (2010) The Pseudomonas aeruginosa Vfr regulator controls global virulence factor expression through cAMP-dependent and -independent mechanisms. J Bacteriol. 192(14): 3553-3564.
Fuchs EL, Brutinel ED, Klem ER, Fehr AR, Yahr TL and Wolfgang MC. (2010) In vitro and in vivo characterization of the Pseudomonas aeruginosa cAMP phosphodiesterase CpdA required for cAMP homeostasis and virulence factor regulation. J Bacteriol. 192(11): 2779-2790.
Fulcher NB, Holliday PM, Klem E, Cann MJ and Wolfgang MC. (2010) The Pseudomonas aeruginosa Chp chemosensory system regulates intracellular cAMP levels by modulating adenylate cyclase activity. Mol Microbiol. 76(4): 889-904.
Orans J, Johnson MDL, Coggan KA, Sperlazza JR, Heiniger RW, Wolfgang MC and Redinbo MR. (2010) Crystal structure analysis reveals Pseudomonas PilY1 as an essential calcium-dependent regulator of bacterial surface motility. Proc Natl Acad Sci USA. 107(11): 1065-1070.
Martino ME, Olsen JC, Fulcher NB, Wolfgang MC, O’Neal WK and Ribeiro CM. (2009) Airway epithelial inflammation-induced endoplasmic reticulum Ca2+ store expansion is mediated by X-box binding protein-1. J Biol Chem. 284(22): 14904-14913.
Cody WL, Pritchett CL, Jones AK, Carterson AJ, Jackson D, Frisk A, Wolfgang MC and Schurr MJ. (2009) Pseudomonas aeruginosa AlgR controls cyanide production in an AlgZ-dependent manner. J Bacteriol. 191(9): 2993-3002.
Tunney MM, Field TR, Moriarty TF, Patrick S, Doering G, Muhlebach MS, Wolfgang MC, Boucher R, Gilpin DF, McDowell A and Elborn JS. (2008) Detection of anaerobic bacteria in high numbers in sputum from patients with cystic fibrosis. Am J Respir Crit Care Med. 177(9): 995-1001.
Naessan CL, Egge-Jacobsen W, Heiniger RW, Wolfgang MC, Aas FE, Rohr A, Winther-Larsen HC and Koomey M. (2008) Genetic and functional analyses of PptA, a phospho-form transferase targeting type IV pili in Neisseria gonorrhoeae. J Bacteriol. 190(1): 387-400.
Winther-Larsen HC, Wolfgang MC, van Putten JP, Roos N, Aas FE, Egge-Jacobsen WM, Maier B and Koomey M. (2007) Pseudomonas aeruginosa Type IV pilus expression in Neisseria gonorrhoeae: effects of pilin subunit composition on function and organelle dynamics. J Bacteriol. 189(18): 6676-6685.
Aas FE, Winther-Larsen HC, Wolfgang M, Frye S, Lovold C, Roos N, van Putten JP and Koomey M. (2007) Substitutions in the N-terminal alpha helical spine of Neisseria gonorrhoeae pilin affects Type IV pilus assembly, dynamics and associated functions. Mol Microbiol. 63(1): 69-85.
Yahr TL and Wolfgang MC. (2006) Transcriptional regulation of the Pseudomonas aeruginosa type III secretion system. Review. Mol Microbiol. 62(3): 631-640.
Soong G, Muir A, Gomez MI, Waks J, Reddy B, Planet P, Singh PK, Kanetko Y, Wolfgang MC, Hsiao YS, Tong L and Prince A. (2006) Bacterial neuraminidase facilitates mucosal infection by participating in biofilm production. J Clin Invest. 116(8): 2297-2305.
Kulasekara BR, Kulasekara HD, Wolfgang MC, Stevens L, Frank DW and Lory S. (2006) Acquisition and evolution of the exoU locus in Pseudomonas aeruginosa. J Bacteriol. 188(11): 4037-4050.
Tart AH, Wolfgang MC and Wozniak DJ. (2005) The alternative sigma factor AlgT represses Pseudomonas aeruginosa flagellum biosynthesis by inhibiting expression of fleQ. J Bacteriol. 187(23): 7955-7962.
Ichikawa JK, English SB, Wolfgang MC, Jackson R, Butte AJ and Lory S. (2005) Genome-wide analysis of host responses to the Pseudomonas aeruginosa Type III secretion system yields synergistic effects. Cell Microbiol. 7(11): 1635-1646.
Winther-Larsen HC, Wolfgang M, Dunham S, van Putten JM, Dorward D, Løvold C, Aas FE and Koomey M. (2005) A conserved set of pilin-like molecules controls type IV pilus dynamics and organelle-associated functions in Neisseria gonorrhoeae. Mol Microbiol. 56(4): 903-917.
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