Timothy L. Yahr

ACTIVE AND PASSIVE IMMUNIZATION WITH THE PSEUDOMONAS V ANTIGEN PROTECTS AGAINST TYPE III INTOXICATION AND LUNG INJURY

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause fatal acute lung infections in critically ill individuals. Damage to the lung epithelium is associated with the expression of toxins that are directly injected into eukaryotic cells through a type III-mediated secretion and translocation mechanism. Here we show that the P. aeruginosa homolog of the Yersinia V antigen, PcrV, is involved in the translocation of type III toxins. Vaccination against PcrV ensured the survival of challenged mice and decreased lung inflammation and injury. Antibodies to PcrV inhibited the translocation of type III toxins.

Exoenzyme S of Pseudomonas aeruginosa is secreted by a type III pathway

Exoenzyme S is an extracellular ADP‐ribosyltransferase of Pseudomonas aeruginosa. Transposon mutagenesis of P. aeruginosa 388 was used to identify genes required for exoenzyme S production. Five Tn5 Tc insertion mutants were isolated which exhibited an exoenzyme S‐deficient phenotype (388::Tn5 Tc 469, 550, 3453, 4885, and 5590). Mapping experiments demonstrated that 388::Tn5 Tc 3453, 4885, and 5590 possessed insertions within a 5.0 kb EcoRI fragment that is not contiguous with the exoenzyme S trans‐regulatory operon. 388::Tn5 Tc 469 and 550 mapped to a region downstream of the trans‐regulatory operon which has been previously shown to contain a promoter region that is co‐ordinately regulated with exoenzyme S synthesis. Nucleotide sequence analysis of a 7.2 kb region flanking the 388::Tn5 Tc 469 and 550 insertions, identified 12 contiguous open reading frames (ORFs). Database searches indicated that the first ORF, ExsD, is unique. The other 11 ORFs demonstrated high homology to the YscB–L proteins of the yersiniaeYop type III export apparatus. RNase‐protection analysis of wild‐type and mutant strains indicated that exsD and pscB–L form an operon. To determine whether ExoS was exported by a type III mechanism, derivatives consisting of internal deletions or lacking amino‐ or carboxy‐terminal residues were expressed in P. aeruginosa. Deletion analyses indicated that the amino‐terminal nine residues are required for ExoS export. Combined data from mutagenesis, regulatory, expression, and sequence analyses provide strong evidence that P. aeruginosa possesses a type III secretion apparatus which is required for the export of exoenzyme S and potentially other co‐ordinately regulated proteins.

Transcriptional regulation of the Pseudomonas aeruginosa type III secretion system

Type III secretion systems (T3SS) function by translocating effector proteins into eukaryotic host cells and are important for the virulence of many Gram‐negative bacterial pathogens. Although the secretion and translocation machineries are highly conserved between different species, each pathogen translocates a unique set of effectors that subvert normal host cell physiology to promote pathogenesis. The uniqueness of each pathogen is further reflected in the diversity of mechanisms used to regulate T3SS gene expression. Pseudomonas aeruginosa utilizes a complex set of signalling pathways to modulate T3SS expression in response to extracellular and intracellular cues. Whereas some pathways are dedicated solely to regulating the T3SS, others co‐ordinately regulate expression of the T3SS with multiple virulence functions on a global scale. Emerging regulatory themes include coupling of T3SS transcription with type III secretory activity, global regulatory control through modulation of cAMP biosynthesis, repression by a variety of stresses, involvement of multiple two component regulatory systems, and an inverse relationship between T3SS expression and multicellular behaviour. Factors controlling activation of T3SS expression likely contribute to the environmental survival of the organism and to the pathogenesis of acute P. aeruginosa infections. Conversely, active repression of the T3SS might contribute to the persistence of chronic infections.

Functional reconstitution of bacterial Tat translocation in vitro

The Tat (twin‐arginine translocation) pathway is a Sec‐independent mechanism for translocating folded preproteins across or into the inner membrane of Escherichia coli. To study Tat translocation, we sought an in vitro translocation assay using purified inner membrane vesicles and in vitro synthesized substrate protein. While membrane vesicles derived from wild‐type cells translocate the Sec‐dependent substrate proOmpA, translocation of a Tat‐dependent substrate, SufI, was not detected. We established that in vivo overexpression of SufI can saturate the Tat translocase, and that simultaneous overexpression of TatA, B and C relieves this SufI saturation. Using membrane vesicles derived from cells overexpressing TatABC, in vitro translocation of SufI was detected. Like translocation in vivo, translocation of SufI in vitro requires TatABC, an intact membrane potential and the twin‐arginine targeting motif within the signal peptide of SufI. In contrast to Sec translocase, we find that Tat translocase does not require ATP. The development of an in vitro translocation assay is a prerequisite for further biochemical investigations of the mechanism of translocation, substrate recognition and translocase structure.

ExsD is a negative regulator of the Pseudomonas aeruginosa type III secretion regulon

Expression of the Pseudomonas aeruginosa type III secretion system is induced by contact with eukaryotic cells, serum or low Ca2+ concentrations. We report that ExsD, a unique protein, is a negative regulator of the type III regulon. Localization studies indicate that ExsD is not secreted by P. aeruginosa. To determine the role of exsD, a non‐polar deletion was returned to the chromosome by allelic exchange. The ΔexsD mutant is competent for type III secretion and translocation of the ExoU cytotoxin to eukaryotic host cells. To examine the effect of ExsD on transcription, lacZ transcriptional reporter fusions were integrated into the chromosome. Promoters controlling transcription of genes encoding the type III secretory, regulatory and effector proteins demonstrated significant derepression in the ΔexsD background. Expression of ExsD from a multicopy plasmid completely repressed transcription of the regulon. Although a mutant in pscC, encoding a structural component of the type III translocase, is repressed for expression of the regulon, a ΔexsD, pscC::Ω double mutant is derepressed. Bacterial two‐hybrid data indicate that ExsD binds the transcriptional activator of the regulon, ExsA. We conclude that ExsD is a negative regulator and propose that ExsD functions as an ExsA antiactivator to regulate transcription of the regulon.

A novel anti‐anti‐activator mechanism regulates expression of the Pseudomonas aeruginosa type III secretion system

Expression of the Pseudomonas aeruginosa type III secretion system (TTSS) is coupled to the secretion status of the cells. Environmental signals such as calcium depletion activate the type III secretion channel and, as a consequence, type III gene transcription is derepressed. Two proteins, ExsA and ExsD, were shown previously to play a role in coupling transcription to secretion. ExsA is an activator of TTSS gene transcription, and ExsD is an anti‐activator of ExsA. In the absence of environmental secretion cues, ExsD binds ExsA and inhibits transcription. Here, we describe the characterization of ExsC as an anti‐anti‐activator of TTSS expression. Transcription of the TTSS is repressed in an exsC mutant and is derepressed upon ExsC overexpression. The dependence on exsC for transcription is relieved in the absence of exsD, suggesting that ExsC and ExsD function together to regulate transcription. Consistent with this idea, ExsC interacts with ExsD in bacterial two‐hybrid and co‐purification assays. We propose a model in which the anti‐anti‐activator (ExsC) binds to and sequesters the anti‐activator (ExsD) under low Ca2+ conditions, freeing ExsA and allowing for transcription of the TTSS. The P. aeruginosa system represents the first example of an anti‐activator/anti‐anti‐activator pair controlling transcription of a TTSS.

Evaluating the oligomeric state of SecYEG in preprotein translocase

SecA insertion and deinsertion through SecYEG drive preprotein translocation at the Escherichia coli inner membrane. We present three assessments of the theory that oligomers of SecYEG might form functional translocation sites. (i) Formaldehyde cross‐ linking of translocase reveals cross‐links between SecY, SecE and SecG, but not higher order oligomers. (ii) Cross‐linking of membranes containing unmodified SecE and hemagglutinin‐tagged SecE (SecEHA) reveals cross‐links between SecY and SecE and between SecY and SecEHA. However, anti‐HA immunoprecipitates contain neither untagged SecE nor SecY cross‐linked to SecE. (iii) Membranes containing similar amounts of SecE and SecEHA were saturated with translocation intermediate (I29) and detergent solubilized. Anti‐HA immunoprecipitation of I29 required SecYEHAG and SecA, yet untagged SecE was not present in this translocation complex. Likewise, anti‐HA immunoprecipitates of membranes containing equal amounts of SecY and SecYHA were found to contain SecYHA but not SecY. Both immunoprecipitates contain more moles of I29 than of the untagged subunit, again suggesting that translocation intermediates are not engaged with multiple copies of SecYEG. These studies suggest that the active form of preprotein translocase is monomeric SecYEG.

Control of gene expression by type III secretory activity

The bacterial flagellum and the highly related injectisome (or needle complex) are among the most complicated multi-protein structures found in Gram-negative microorganisms. The assembly of both structures is dependent upon a type III secretion system. An interesting regulatory feature unique to these systems is the coordination of gene expression with type III secretory activity. This means of regulation ensures that secretion substrates are expressed only when required during the assembly process or upon completion of the fully functional structure. Prominent within the regulatory scheme are secreted proteins and type III secretion chaperones that exert effects on gene expression at the transcriptional and post-transcriptional levels. Although the major structural components of the flagellum and injectisome systems are highly conserved, recent studies reveal diversity in the mechanisms used by secretion substrates and chaperones to control gene expression.

Glucose Depletion in the Airway Surface Liquid Is Essential for Sterility of the Airways

Diabetes mellitus predisposes the host to bacterial infections. Moreover, hyperglycemia has been shown to be an independent risk factor for respiratory infections. The luminal surface of airway epithelia is covered by a thin layer of airway surface liquid (ASL) and is normally sterile despite constant exposure to bacteria. The balance between bacterial growth and killing in the airway determines the outcome of exposure to inhaled or aspirated bacteria: infection or sterility. We hypothesized that restriction of carbon sources –including glucose– in the ASL is required for sterility of the lungs. We found that airway epithelia deplete glucose from the ASL via a novel mechanism involving polarized expression of GLUT-1 and GLUT-10, intracellular glucose phosphorylation, and low relative paracellular glucose permeability in well-differentiated cultures of human airway epithelia and in segments of airway epithelia excised from human tracheas. Moreover, we found that increased glucose concentration in the ASL augments growth of P. aeruginosa in vitro and in the lungs of hyperglycemic ob/ob and db/db mice in vivo. In contrast, hyperglycemia had no effect on intrapulmonary bacterial growth of a P. aeruginosa mutant that is unable to utilize glucose as a carbon source. Our data suggest that depletion of glucose in the airway epithelial surface is a novel mechanism for innate immunity. This mechanism is important for sterility of the airways and has implications in hyperglycemia and conditions that result in disruption of the epithelial barrier in the lung.

Intrinsic and extrinsic regulation of type III secretion gene expression in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that is particularly problematic in the healthcare setting where it is a frequent cause of pneumonia, bloodstream, and urinary tract infections. An important determinant of P. aeruginosa virulence is a type III secretion system (T3SS). T3SS-dependent intoxication is a complex process that minimally requires binding of P. aeruginosa to host cells, injection of the cytotoxic effector proteins through the host cell plasma membrane, and induction of T3SS gene expression. The latter process, referred to as contact-dependent expression, involves a well-characterized regulatory cascade that activates T3SS gene expression in response to host cell contact. Although host cell contact is a primary activating signal for T3SS gene expression, the involvement of multiple membrane-bound regulatory systems indicates that additional environmental signals also play a role in controlling expression of the T3SS. These regulatory systems coordinate T3SS gene expression with many other cellular activities including motility, mucoidy, polysaccharide production, and biofilm formation. The signals to which the organism responds are poorly understood but many seem to be coupled to the metabolic state of the cell and integrated within a master circuit that assimilates informational signals from endogenous and exogenous sources. Herein we review progress toward unraveling this complex circuitry, provide analysis of the current knowledge gaps, and highlight potential areas for future studies. Complete understanding of the regulatory networks that control T3SS gene expression will maximize opportunities for the development of strategies to treat P. aeruginosa infections.